Dedifferentiated, programmable stem cells of monocytic origin, and their production and use

ABSTRACT

The invention relates to the production of adult dedifferentiated, programmable stem cells from human monocytes by cultivation of monocytes in a culture medium which contains M-CSF and IL-3. The invention further relates to pharmaceutical preparations, which contain the dedifferentiated, programmable stem cells and the use of these stem cells for the production of target cells and target tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.10/372,657 filed Feb. 25, 2003. This application claims priority under35 U.S.C. §§ 119 and 120 to German application No. 102 14 095.2 filedMar. 28, 2002, International Application No. PCT/EP03/02121 filed Feb.25, 2003, and U.S. patent application Ser. No. 10/372,657 filed Feb. 25,2003, each of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] The term “stem cells” designates cells which (a) have thecapability of self-renewal and (b) the capability to form at least oneand often a number of specialized cell types due to their asymmetricaldivision capability (cf. Donovan, P. J., Gearhart, J., Nature 414: 92-97(2001)). The term “pluripotent” designates stem cells, which canessentially be differentiated into all possible cell types of the humanand animal body. Such stem cells have hitherto only been obtainable fromembryonic tissue or embryonic carcinoma (testicular tumor) (cf. Donovan,P. J., Gearhart, J., loc cit). The use of embryonic stem cells has beenthe subject of extensive public discussion, especially in Germany, andis regarded as extremely problematical. Besides the ethical and legalproblems connected with embryonic stem cells, the therapeutic use ofsuch cells also comes up against difficulties. By nature, embryonic stemcells are obtained from donor organisms, which are heterologousvis-à-vis the potential recipients of differentiated cells or tissue(hereafter referred to as somatic target cells or target tissue)developed from these cells. It is therefore to be expected, that suchtarget cells will trigger an immediate immunological response in thepotential recipients in the form of rejection.

[0003] Stem cells can be also isolated from different tissues of adult,i.e., from differentiated individuals. Such stem cells are referred toin the state of the art as “multipotent adult stem cells”. In the bodythey play a role in tissue regeneration and homeostasis. The essentialdifference between embryonic pluripotent stem cells and adultmultipotent stem cells lies in the number of differentiated tissues,which can be obtained from the respective cells. Presumably, this is dueto the fact that pluripotent stem cells come from sperm cells, or fromcells which can produce sperm, while adult multipotent stem cells comefrom the body or soma of adult individuals (cf. Donovan, P. J.,Gearhart, J., loc cit, Page 94), which are not capable of spermproduction.

[0004] The actual problems relating to the obtaining and use of adultstem cells however lie in the rarity of these cells. Thus, in the bonemarrow, stem cells are present only in the ratio of 1:10,000, in theperipheral blood of 1:250,000 and in the liver in the ratio of1:100,000. Obtaining such stem cells is therefore very expensive andstressful for the patient. In addition the generation of large cellquantities, as required for clinical therapy, has scarcely been possiblehitherto at reasonable expense.

[0005] This is contrasted by a constantly increasing need forpossibilities for treatment of destroyed tissue in the form of “tissueengineering” or as cell therapy, within the framework of which skin-,muscle-, heart muscle-, liver-, islet-, nerve-, neurone-, bone-,cartilage-, endothelium- and fat cells etc. are to be replaced.

[0006] In this connection, the foreseeable development of the age anddisease profile of the population in the western world is decisive,leading to the expectation of a drastic turning point in the next 10years in the health and care sector of the western European population,including the USA and Canada. In the Federal Republic of Germany alone,the demographic development suggests a 21%-growth in population in the45-64 year-old age group by 2015, and a 26%-growth in the over-65 agegroup. This is bound to result in a change in patient structure and inthe spectrum of diseases requiring treatment. Predictably, diseases ofthe cardio-circulatory system (high pressure, myocardial infarction),vascular diseases due to arteriosclerosis and metabolic diseases,metabolic diseases such an diabetes mellitus, diseases at livermetabolism, kidney diseases as well as diseases of the skeletal systemcaused by age-related degeneration, and degenerative diseases of thecerebrum caused by neuronal and glial cell losses will increase andrequire innovative treatment concepts.

[0007] These facts explain the immense national and internationalresearch and development efforts by the specialists involved, to obtainstem cells which can be programmed into differentiated cells typical oftissue (liver, bone, cartilage, muscle, skin etc.).

[0008] The problem underlying the invention therefore resides in makingavailable adult stem cells, the generation of which gives rise to noethical and/or legal problems, which are rapidly available for theplanned therapeutic use in the quantities required for this, and atjustifiable production costs, and which, when used as “cellulartherapeutics” give rise to no side effects—or none worth mentioning—interms of cellular rejection and induction of tumors, particularlymalignant tumors, in the patient in question.

SUMMARY OF THE INVENTION

[0009] The present invention provides a method for producing humandedifferentiated programmable stem cells using M-CSF and IL-3.

[0010] The present invention includes and provides a process for theproduction of dedifferentiated, programmable stem cells of humanmonocytic origin, comprising (a) isolating the monocytes from humanblood; (b) propagating the monocytes in a culture medium, which containscellular growth factor M-CSF; (c) simultaneously cultivating themonocytes with or subsequently to step (b) in a culture mediumcomprising IL-3; and (d) obtaining human adult dedifferentiatedprogrammable stem cells by separating from culture medium.

[0011] The present invention includes and provides a process for theproduction of dedifferentiated, programmable stem cells of humanmonocytic origin, comprising (a) providing human monocytes; (b)propagating the monocytes in a culture medium, which contains cellulargrowth factor M-CSF; (c) simultaneously cultivating the monocytes withor subsequently to step (b) in a culture medium comprising IL-3; and (d)obtaining human adult dedifferentiated programmable stem cells byseparating from culture medium.

[0012] The present invention includes and provides a dedifferentiated,programmable stem cell of human monocytic origin, wherein the cell ischaracterized by exhibiting a CD14 antigen and an antigen selected fromthe group consisting of CD90, CD117, CD123 and CD135.

[0013] The present invention includes and provides a dedifferentiated,programmable stem cell of human monocytic origin, wherein the cell ischaracterized by exhibiting a CD14 antigen and a CD123 antigen.

[0014] The present invention includes and provides a dedifferentiated,programmable stem cell of human monocytic origin, wherein the cell ischaracterized by exhibiting a CD14 antigen and a CD135 antigen.

[0015] The present invention includes and provides a dedifferentiated,programmable stem cell of human monocytic origin, wherein the cell ischaracterized by exhibiting a CD14 antigen, a CD123 antigen and a CD135antigen.

[0016] The present invention includes and provides a dedifferentiated,programmable stem cell of human monocytic origin manufactured by aprocess comprising (a) isolating monocytes from human blood; (b)propagating monocytes in a culture medium, which contains cellulargrowth factor M-CSF; (c) simultaneously cultivating monocytes with orsubsequently to step (b) in a culture medium comprising IL-3; and (d)obtaining human adult dedifferentiated programmable stem cells byseparating from culture medium.

[0017] The present invention includes and provides a pharmaceuticalcomposition comprising a dedifferentiated, programmable stem cell ofhuman monocytic origin, wherein the cell is characterized by exhibitinga CD14 antigen and an antigen selected from the group consisting ofCD90, CD117, CD123 and CD135.

[0018] The present invention includes and provides a pharmaceuticalcomposition comprising a dedifferentiated, programmable stem cell ofhuman monocytic origin, wherein the cell is characterized by exhibitinga CD14 antigen and a CD135 antigen.

[0019] The present invention includes and provides a pharmaceuticalcomposition comprising a dedifferentiated, programmable stem cell ofhuman monocytic origin, wherein the cell is characterized by exhibitinga CD14 antigen and a CD123 antigen.

[0020] The present invention includes and provides a pharmaceuticalcomposition comprising a dedifferentiated, programmable stem cell ofhuman monocytic origin, wherein the cell is characterized by exhibitinga CD14 antigen, a CD123 antigen and a CD135 antigen.

[0021] The present invention includes and provides a method of producingtarget cells from dedifferentiated, programmable stem cells of humanmonocytic origin comprising (a) obtaining desired target cells from atarget tissue; (b) incubating the desired target cells in a suitableculture medium; and (c) providing supernatant from the culture mediumafter incubation with the desired target cells to dedifferentiated,programmable stem cells of human monocytic origin that are characterizedby exhibiting a CD14 antigen and an antigen selected from the groupconsisting of CD90, CD117, CD123 and CD135 to differentiate said stemcells of human monocytic origin into target cells.

[0022] The present invention includes and provides a method of producingtarget cells from dedifferentiated, programmable stem cells of humanmonocytic origin comprising (a) obtaining desired target cells from atarget tissue; (b) incubating the desired target cells in a suitableculture medium; and (c) providing supernatant from the culture mediumafter incubation with the desired target cells to dedifferentiated,programmable stem cells of human monocytic origin that are characterizedby exhibiting a CD14 and a CD135 antigen to differentiate said stemcells of human monocytic origin into target cells.

[0023] The present invention includes and provides a method of producingtarget cells from dedifferentiated, programmable stem cells of humanmonocytic origin comprising (a) obtaining desired target cells from atarget tissue; (b) incubating the desired target cells in a suitableculture medium; and (c) providing supernatant from the culture mediumafter incubation with the desired target cells to dedifferentiated,programmable stem cells of human monocytic origin that are characterizedby exhibiting a CD14 antigen and a CD123 antigen to differentiate saidstem cells of human monocytic origin into target cells.

[0024] The present invention includes and provides a method of producingtarget cells from dedifferentiated, programmable stem cells of humanmonocytic origin comprising (a) obtaining desired target cells from atarget tissue; (b) incubating the desired target cells in a suitableculture medium; and (c) providing supernatant from the culture mediumafter incubation with the desired target cells to dedifferentiated,programmable stem cells of human monocytic origin that are characterizedby exhibiting a CD14 antigen, a CD123 antigen and a CD135 antigen todifferentiate said stem cells of human monocytic origin into targetcells.

[0025] According to the present invention, the methods of producingtarget cells from dedifferentiated, programmable stem cells of humanmonocytic origin thus start with the isolation of desired target cells(step a), i.e. the isolation of differentiated cells of the cell typewhich is to be produced using the dedifferentiated, programmable stemcells. The differentiated target cells can be incubated in a cellculture medium (step b). Supernatant from the cell culture medium of thedifferentiated target cells can be used to differentiate stem cells ofhuman monocytic origin into target cells (c).

[0026] The present invention includes and provides a dedifferentiated,programmable stem cell of human monocytic origin, wherein the cell ischaracterized by the membrane associated monocyte-specific surfaceantigen CD14 and at least one pluripotency marker selected from thegroup consisting of CD117, CD123 and CD135.

[0027] The present invention includes and provides a dedifferentiated,programmable stem cell preparation comprising a dedifferentiated,programmable stem cell of human monocytic origin of the presentinvention in a suitable medium.

[0028] The present invention includes and provides a method for treatingliver cirrhosis using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

[0029] The present invention includes and provides a method of making apharmaceutical composition for treating liver cirrhosis by preparing acomposition comprising dedifferentiated programmable stem cells of thepresent invention.

[0030] The present invention includes and provides a method for treatingpancreatic insufficiency using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

[0031] The present invention includes and provides a method of making apharmaceutical composition for treating pancreatic insufficiency bypreparing a composition comprising dedifferentiated programmable stemcells of the present invention.

[0032] The present invention includes and provides a method for treatingacute or chronic kidney failure using a pharmaceutical compositioncomprising dedifferentiated programmable stem cells of the presentinvention.

[0033] The present invention includes and provides a method of making apharmaceutical composition for treating acute or chronic kidney failureby preparing a composition comprising dedifferentiated programmable stemcells of the present invention.

[0034] The present invention includes and provides a method for treatinghormonal underfunctioning using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

[0035] The present invention includes and provides a method of making apharmaceutical composition for treating hormonal underfunctioning bypreparing a composition comprising dedifferentiated programmable stemcells of the present invention.

[0036] The present invention includes and provides a method for treatingcardiac infarction using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

[0037] The present invention includes and provides a method of making apharmaceutical composition for treating cardiac infarction by preparinga composition comprising dedifferentiated programmable stem cells of thepresent invention.

[0038] The present invention includes and provides a method for treatingpulmonary embolisms using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

[0039] The present invention includes and provides a method of making apharmaceutical composition for treating pulmonary embolisms by preparinga composition comprising dedifferentiated programmable stem cells of thepresent invention.

[0040] The present invention includes and provides a method for thetreatment of stroke using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

[0041] The present invention includes and provides a method of making apharmaceutical composition for the treatment of stroke by preparing acomposition comprising dedifferentiated programmable stem cells of thepresent invention.

[0042] The present invention includes and provides a method for thetreatment of skin damage using a pharmaceutical composition comprisingdedifferentiated programmable stem cells of the present invention.

[0043] The present invention includes and provides a method of making apharmaceutical composition for the treatment of skin damage by preparinga composition comprising dedifferentiated programmable stem cells of thepresent invention.

[0044] The present invention includes and provides differentiated,isolated, somatic target cells and/or target tissue, characterized bythe membrane-associated surface antigen CD14. Such cells can beobtained, for example, without limitation, by reprogramming the stemcells according to a method of the present invention.

[0045] The present invention includes and provides differentiated,isolated, somatic target cells and/or target tissue characterized by themembrane-associated surface antigen CD14 where the target cells and/ortarget tissue is selected from the group consisting of adipocytes,neurons, glia cells, endothelial cells, keratinocytes, hepatocytes andislet cells.

[0046] The present invention includes and provides differentiated,isolated, somatic target cells and/or target tissue, characterized bythe membrane-associated surface antigen CD14, further comprising atransfected gene. Such cells can be obtained, for example, withoutlimitation, by reprogramming the stem cells according to a method of thepresent invention.

[0047] The present invention includes and provides implantable materialscoated with the dedifferentiated, programmable stem cells includingdifferentiated, isolated, somatic target cells and/or target tissue,obtained by reprogramming the stem cells according to a method of thepresent invention.

[0048] The present invention includes and provides implantable materialsthat are prostheses, including those selected from the group consistingof cardiac valves, vessel prostheses, bone and joint prostheses, coatedwith the dedifferentiated, programmable stem cells includingdifferentiated, isolated, somatic target cells and/or target tissue,obtained by reprogramming the stem cells according to a method of thepresent invention.

[0049] The present invention includes and provides implantable materialsthat are artificial and/or biological carrier materials comprising thededifferentiated, programmable stem cells including differentiated,isolated, somatic target cells and/or target tissue, obtained byreprogramming the stem cells according to a method of the presentinvention.

[0050] The present invention includes and provides implantable materialsthat are bags or chambers for introduction into the human bodycontaining differentiated, isolated, somatic target cells and/or targettissue, obtained by reprogramming the stem cells according to a methodof the present invention.

[0051] The present invention includes and provides implantable materialsthat are bags or chambers, containing islet cells of the presentinvention, for introduction into the human body containingdifferentiated, isolated, somatic target cells and/or target tissue,obtained by reprogramming the stem cells according to a method of thepresent invention for the production of a pharmaceutical construct foruse as an artificial islet cell portchamber for the supply of insulin.

[0052] The present invention includes and provides implantable materialsthat are bags or chambers, containing adipocytes of the presentinvention, for introduction into the human body containingdifferentiated, isolated, somatic target cells and/or target tissue,obtained by reprogramming the stem cells according to a method of thepresent invention for the production of a pharmaceutical construct,which contains artificial polymers filled with adipocytes, for breastconstruction after surgery and for use in the case of plastic and/orcosmetic correction.

[0053] The present invention includes and provides implantable materialsthat are semi-permeable port chamber systems comprising thededifferentiated, programmable stem cells including differentiated,isolated, somatic target cells and/or target tissue, obtained byreprogramming the stem cells according to a method of the presentinvention.

[0054] The present invention includes and provides implantable materialsthat are semi-permeable port chamber systems comprising thededifferentiated, programmable stem cells including differentiated,isolated, somatic target cells and/or target tissue, obtained byreprogramming the stem cells according to a method of the presentinvention for the production of a pharmaceutical construct for in vivotreatment of endocrine, metabolic or hemostatic diseases.

DETAILED DESCRIPTION

[0055] The invention relates to adult dedifferentiated programmable stemcells derived from human monocytes, as well as their production and usefor the production of body cells and tissues. According to aparticularly preferred embodiment of the invention these cells areautologous human stem cells, i.e., the cell of monocytic origin comesfrom the patient who is to be treated with the stem cell produced fromthe original cell and/or with the body cells produced from this stemcell.

[0056] According to the invention this problem is solved by theproduction of dedifferentiated programmable cells from human monocyteswhich, for the purposes of the invention, are referred to hereafter as“stem cells”. The term “dedifferentiation” is familiar to the personskilled in the relevant art, cf. for Weissman I. L., Cell 100: 157-168,FIG. 4, (2000). It signifies the regression of an adult, alreadyspecialized (differentiated) body cell to the status of a stem cell,i.e., of a cell, which in turn can be transferred (programmed) into anumber of cell types. Surprisingly, it has been demonstrated that theprocess according to the invention leads to the dedifferentiation ofmonocytes. The stem cells produced in this way can be transformed(programmed) into a large number of different target cells/targettissue, cf. examples. The stem cells according to the invention express,in addition to the CD14 surface antigen characteristic of differentiatedmonocytes, at least one, preferably two or three, of the typicalpluripotency markers CD90, CD117, CD123 and CD135. In a particularlypreferred manner, the stem cells produced according to the inventionexpress the CD14 surface antigen as well as the four pluripotencymarkers CD90, CD117, CD123 and CD135, cf. Example 2, Table 1.Preferably, the stem cells of the invention express the membraneassociated monocyte-specific surface antigen CD14 and at least onepluripotency markers selected from the group consisting of CD117, CD123and CD135. More preferably, the stem cells of the invention carry theCD14 antigen in combination with at least the pluripotency marker CD123and/or CD135. Less than 3%, preferably less than 1% of the stem cellsaccording to the invention express the CD34 antigen. Most preferably,none of the stem cells of the invention express the CD34 antigen. Inthis way, for the first time adult stem cells are made available, whichcan within a short time be reprogrammed into preferably autologoustissues.

[0057] The generation of the stem cells according to the invention iscompletely harmless to the patient and—in the case of autologoususe—comparable to own blood donation. The quantity of stem cells (10⁸ to10⁹ cells) required for the usual therapy options (see above) can bemade available cost-effectively within 10 to 14 days after the blood istaken. In addition the cell product provided for the therapy, in thecase of autologous use, does not give rise to any immunological problemin terms of cell rejection, as cells and recipient are preferablygenetically identical.

[0058] The stem cells according to the invention have also proved to berisk-free in animal experimentation and in culture with regard to givingrise to malignancy, a result which is only to be expected due to thecell of monocytic origin, from which the stem cells according to theinvention derive.

[0059] In one aspect, steps of the process according to the inventionfor the production of dedifferentiated programmable stem cells of humanmonocytic origin comprise:

[0060] (a) Isolation of monocytes from human blood;

[0061] (b) Propagating the monocytes in a suitable culture vesselcontaining cell culture medium, which contains themacrophage-colony-stimulating factor (hereafter referred to as M-CSF);and

[0062] (c) Cultivating the monocytes in the presence of interleukin-3(IL-3); and

[0063] (d) Obtaining the human dedifferentiated programmable stem cells,by separating the cells from the culture medium.

[0064] According to a particularly preferred embodiment of the process,M-CSF and IL-3 are simultaneously added to the cell culture medium inStep b).

[0065] It is however also possible, initially only to add M-CSF to thecell culture medium in Step b) in order to cause the monocytes topropagate, and to add IL-3 to the cell culture medium subsequently.

[0066] Finally the process in Step b) can also be carried out in such away that the monocytes are initially propagated in a cell culture mediumcontaining only M-CSF, then the medium is separated from the cells and asecond cell culture medium is then used, which contains IL-3.

[0067] According to a preferred embodiment of the invention the culturemedium of Step b) is separated from the cells attached to the bottom ofthe culture vessel and the human, dedifferentiated, programmable stemcells are obtained by detaching the cells from the bottom and byisolating the cells.

[0068] According to a preferred embodiment of the invention the cellsare further cultivated in the presence of a sulfur compound. Thecultivation can be carried out in a separate process step which followsthe cultivation Step b) illustrated above. It can however also becarried out in Step b), by further adding the sulfur compound to theculture medium, preferably already at the start of the cultivation.

[0069] The process according to the invention surprisingly leads to thededifferentiation of the monocytes, wherein the adult stem cellsresulting from the dedifferentiation, besides the CD14 surface antigentypical of the differentiated monocytes, also express at least one ormore, preferably all of the pluripotency markers CD90, CD117, CD123 andCD135 (cf. Table 1). Preferably, the stem cells of the invention expressthe membrane associated monocyte-specific surface antigen CD14 and atleast one pluripotency markers selected from the group consisting ofCD117, CD123 and CD135. More preferably, the stem cells of the inventioncarry the CD14 antigen in combination with at least the pluripotencymarker CD123 and/or CD135. Less than 3%, preferably less than 1% of thestem cells according to the invention express the CD34 antigen. Mostpreferably, none of the stem cells of the invention express the CD34antigen. The expression of the respective markers (surface antigens) canbe proved by means of commercially available antibodies with specificityagainst the respective antigens to be detected, using standard immunoassay procedures, cf. Example 2.

[0070] As the cells, during the propagation and dedifferentiationprocess, adhere to the bottom of the respective culture vessel, it isnecessary to separate the cells from the culture medium from Step b) andto detach them from the bottom after completion of thededifferentiation. According to a preferred embodiment of the inventionthe cell culture supernatant is discarded before the detaching of thecells adhering to the bottom and subsequently, the adhering cells arepreferably rinsed with fresh culture medium. Following the rinsing,fresh culture medium is again added to the cells adhering to the bottom,and the step of releasing the cells from the bottom then follows (cf.Example 13).

[0071] According to a preferred embodiment the cells are brought intocontact with a biologically well-tolerated organic solvent, at the endof Step c) and before Step d). A biologically well-tolerated organicsolvent can be an alcohol with 1-4 carbon atoms, the use of ethanolbeing preferred.

[0072] In a further embodiment, at the end of Step c) and before Step d)the cells are brought into contact with the vapor phase of thebiologically well-tolerated organic solvent.

[0073] The detaching can moreover also be carried out mechanically,however, an enzymatic detaching process is preferred, for example withtrypsin.

[0074] The dedifferentiated programmable stem cells obtained in thisway, floating freely in the medium, can either be directly transferredto a reprogramming process, or kept in the culture medium for a fewdays; in the latter case, a cytokine or LIF (leukemia inhibitory factor)is preferably added to the medium, in order to avoid premature loss ofthe programmability (cf. Donovan, P. J., Gearhart, J., loc. cit., Page94). Finally the cells can be deep-frozen for storage purposes withoutloss of programmability.

[0075] The stem cells according to the invention differ from thepluripotent stem cells of embryonic origin known hitherto and from theknown adult stem cells from different tissues, in that besides themembrane-associated monocyte-specific CD14 surface antigen, they carryat least one pluripotency marker from the group consisting of CD90,CD117, CD123 and CD135 on their surface. Preferably, the stem cells ofthe invention carry the membrane associated monocyte-specific surfaceantigen CD14 and at least one pluripotency markers selected from thegroup consisting of CD117, CD123 and CD135. More preferably, the stemcells of the invention carry the CD14 antigen in combination with atleast the pluripotency marker CD123 and/or CD135. Less than 3%,preferably less than 1% of the stem cells according to the inventionexpress the CD34 antigen. Most preferably, none of the stem cells of theinvention express the CD34 antigen.

[0076] The stem cells produced using the process according to theinvention can be reprogrammed into any body cells. Processes forreprogramming stem cells are known in the state of the art, cf. forexample Weissman I. L., Science 287: 1442-1446 (2000) and Insight ReviewArticles Nature 414: 92-131 (2001), and the handbook “Methods of TissueEngineering”, Eds. Atala, A., Lanza, R. P., Academic Press, ISBN0-12-436636-8; Library of Congress Catalog Card No. 200188747.

[0077] The differentiated isolated somatic target cells and/or thetarget tissue obtained by reprogramming of the stem cells according tothe invention moreover carry the membrane-associated CD14differentiation marker of the monocytes. Additionally, less than 3%,preferably less than 1% of these somatic target cells and/or thesetarget tissues according to the invention express the CD34 antigen. Mostpreferably, none of these cells or tissues express the CD34 antigen. Asshown in Example 11, hepatocytes which are derived from the stem cellsaccording to the invention, express the CD14 surface marker which istypical of monocytes, while at the same time they produce the proteinalbumin, which is typical of hepatocytes. The hepatocytes derived fromthe stem cells according to the invention can therefore be distinguishedfrom natural hepatocytes. In the same way, the membrane-associated CD14surface marker was detected on insulin-producing cells, which werederived from the stem cells according to the invention (Example 9).

[0078] In one embodiment of the invention the dedifferentiated,programmable stem cells are used for the in-vitro production of targetcells and target tissue (cf. Examples). Accordingly the inventionprovides methods of producing target cells from dedifferentiated,programmable stem cells of human monocytic origin which methods comprisethe isolation of desired target cells as a first step (a), i.e. theisolation of differentiated cells of the cell type which is to beproduced using the dedifferentiated, programmable stem cells. Thedifferentiated desired target cells can be incubated in a cell culturemedium (as a second step, b). Supernatant from the cell culture mediumof the differentiated target cells can be used to differentiate stemcells of human monocytic origin into target cells (in a third step, c).Illustrated methods are set forth in further detail in Example 6(adipocytes), Example 7 (hepatocytes) and Example 8 (keratinocytes).Therefore, differentiated, isolated tissue cells, which are obtained bydifferentiation (reprogramming) of the stem cells according to theinvention, and which carry the membrane-associated CD14 surface antigen,are also subject of the present invention.

[0079] The stem cells according to the invention are preferably simplyand reliably differentiated in vitro into desired target cells, such asfor example adipocytes (cf. Example 6), neurons and glia cells (cf.Example 3), endothelial cells (cf. Example 5), keratinocytes (cf.Example 8), hepatocytes (cf. Example 7) and islet cells (islet ofLangerhans, cf. Example 9), by growing the stem cells in a medium whichcontains the supernatant of the culture medium, in which the respectivetarget cells and/or fragments thereof have been incubated (cf. Examples6 to 8). This supernatant is referred to hereafter as“target-cell-conditioned medium”.

[0080] For the differentiation (reprogramming) of the dedifferentiatedstem cells according to the invention the following procedure cantherefore be followed, in which:

[0081] a) tissue which contains or consists of the desired cells iscrushed;

[0082] b) the desired tissue cells and/or fragments of these areobtained;

[0083] c) the desired cells and/or fragments of these are incubated in asuitable culture medium;

[0084] d) the culture medium supernatant is collected during and afterthe incubation as desired-cell-conditioned medium; and

[0085] e) for the reprogramming/differentiation of dedifferentiated stemcells into the desired cells or tissue, the stem cells are grown in thepresence of the desired-cell-conditioned medium.

[0086] Standard cell culture media can be used as culture medium (cf.Examples). The media preferably contain growth factors, such as forexample the epidermal growth factor.

[0087] The incubation of the desired cells and/or fragments of these(“cell pellet”) can be carried out over 5 to 15, preferably 10 days. Thesupernatant, i.e., the desired-cell-conditioned medium is preferablyremoved in each case after 2 to 4 days and replaced by fresh medium. Thesupernatants thus obtained can be filtered under sterile conditionsseparately or pooled and stored at approximately −20° C. or useddirectly for the programming of stem cells. As shown above, theprogramming of the stem cells into the desired target cells is carriedout by growing stem cells in the presence of the medium conditioned withthe respective desired cells (cf. Examples). The growth mediumpreferably additionally contains a desired-cell-specific growth factor,such as for example the “hepatocyte growth factor” or the “keratinocytegrowth factor” (cf. Examples).

[0088] In one embodiment of the invention the dedifferentiated,programmable stem cells according to the invention are used per se forthe production of a pharmaceutical composition for the in-vivoproduction of target cells and target tissue.

[0089] Such pharmaceutical preparations can contain the stem cellsaccording to the invention suspended in a physiologically well-toleratedmedium. Suitable media are for example PBS (phosphate buffered saline)or physiological saline with 20% human albumin solution and the like.

[0090] These pharmaceutical preparations contain vital dedifferentiated,programmable stem cells according to the invention, which have on theirsurface the CD14 surface marker and at least one more of the multipotentstem cell markers CD90, CD117, CD123 and/or CD135, in a quantity of atleast 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50%, preferably 60 or70%, particularly preferably 80 or 90% and extremely preferably 100%,relative to the total number of the cells present in the preparation,and optionally further pharmaceutically well-tolerated adjuvants and/orcarrier substances.

[0091] Stem cell preparations can contain vital dedifferentiated,programmable stem cells according to the invention, which have on theirsurface the CD14 surface marker and at least one more of the pluripotentstem cell markers CD90, CD117, CD123 and/or CD135, in a quantity of atleast 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58 or 59%, preferably at least 60%, relative to the total numberof the cells present in the preparation; cell suspensions in a cellculture- or transport medium well-tolerated by cells, such as e.g., PBSor RPMI etc., or deep-frozen cell preparations in a suitable storagemedium, such as e.g., RPMI with 50% human albumin solution and 10% DMSOare preferred.

[0092] The number of vital cells and hence the proportion of these inthe compositions referred to above, can be determined optically by useof the “Trypan blue dye exclusion technique”, as vital cells can beoptically distinguished from non-vital cells, using this dye.

[0093] As a rule, it will be irrelevant for clinical use, if some of thecells present in the pharmaceutical preparation do not fulfill thecriteria of dedifferentiated, programmable stem cells according to theinvention, provided that a sufficient number of functional stem cells ispresent. It is however also possible to eliminate non-dedifferentiatedcells by means of processes known in the state of the art on the basisof surface markers typical of the dedifferentiated cells according tothe invention in such preparations, so that these contain the desiredcells in essentially pure form. One example of a suitable process is“Immuno magnetic bead sorting”, cf. Romani et al., J. Immunol. Methods196: 137-151 (1996).

[0094] Stem cells further have the capability, of spontaneouslydifferentiating in vivo by direct contact with a cell group of aspecific cell type into cells of this type. Processes for tissueproduction using cells which can be redifferentiated (“tissueengineering”) are known in the state of the art. For example Wang, X. etal. (“Liver repopulation and correction of metabolic liver disease bytransplanted adult mouse pancreatic cells” Am. J. Pathol. 158 (2):571-579 (2001)), have shown that even certain adult cells of thepancreas in mice are able to transform, in FAH- (fumaroylaceto-acetatehydrolase)-deficient mice, into hepatocytes, which can fully compensatefor the metabolic defect in these animals. A further example is theexperiments of Lagasse et al., “Purified hematopoietic stem cells candifferentiate into hepatocytes in vivo”, Nature Medicine, 6 (11):1229-1234 (2000). The authors have shown that hematopoietic stem cellsfrom bone marrow were able, after in-vivo transfer into FAH-deficientmice, to transform into hepatocytes, which could then compensate for themetabolic defect; see also the review by Grompe M., “Therapeutic LiverRepopulation for the Treatment of Metabolic Liver Diseases” Hum. Cell,12: 171-180 (1999).

[0095] Particularly preferable forms of application for the in-vivodifferentiation of the dedifferentiated stem cells according to theinvention are injection, infusion or implantation of the stem cells intoone specific cell association in the body, in order to allow for thestem cells to differentiate there, by direct contact with the cellassociation, into cells of this cell type. For injection or infusion thecells can be administered in PBS (phosphate buffered saline).

[0096] Preferred examples of the relevant indications in this connectionare: cirrhosis of the liver, pancreatic insufficiency, acute or chronickidney failure, hormonal under-functioning, cardiac infarction,pulmonary embolism, stroke and skin damage.

[0097] Therefore preferred embodiments of the invention are the use ofthe dedifferentiated, programmable stem cells for the production ofdifferent pharmaceutical compositions for the treatment of cirrhosis ofthe liver, pancreatic insufficiency, acute or chronic kidney failure,hormonal under-functioning, cardiac infarction, pulmonary embolism,stroke and skin damage.

[0098] For the therapeutic use of the target cells obtainable from thestem cells according to the invention, a number of concepts areavailable (see above Science 287: 1442-1446 (2000) and Nature 414:92-131 (2001)).

[0099] A further preferred application concerns the injection of thededifferentiated stem cells according to the invention into theperitoneum, so that they differentiate there, due to the influence ofthe cells surrounding them, into peritoneal cells. In the case ofperitoneal dialysis of patients with kidney insufficiency, these cellscan take over a kidney function via their semi-permeable membrane andgive off kidney dependent waste substances into the peritoneum fromwhere these are removed via the dialysate.

[0100] Therefore, also the differentiated, isolated, somatic targetcells and/or target tissue, which are obtained by reprogramming of thestem cells and are characterized by the membrane-associated CD14 antigenare subject of the invention. These somatic target cells and/or targettissue preferably contain adipocytes, neurons and glia cells,endothelial cells, keratinocytes, hepatocytes and islet cells.

[0101] However the cells can also be introduced directly into the organto be reconstituted. The introduction can be carried out via matrixconstructions which are coated with corresponding differentiated cellsor cells capable of differentiation. The matrix constructions are as arule biodegradable, so that they disappear out of the body while thenewly introduced cells grow together with the cells present. From thispoint of view, for example cellular, preferably autologous transplantsin the form of islet cells, hepatocytes, fat cells, skin cells, muscles,cardiac muscles, nerves, bones, endocrine cells etc. come underconsideration for restitution for example after partial surgicalresection of an organ, for repair for example after trauma or forsupportive use, for example in the case of lacking or insufficient organfunction.

[0102] The stem cells according to the invention and target cellsobtained from them can further be used to coat implantable materials, inorder to increase biocompatibility. Therefore, also implantablematerials, which are coated with the dedifferentiated, programmable stemcells or the somatic target cells and/or target tissue are subject ofthe invention. According to one embodiment of the invention theseimplantable materials are prostheses. In particularly preferredembodiments these prostheses are cardiac valves, vessel prostheses,bone- and joint prostheses.

[0103] The implantable materials can also be artificial and/orbiological carrier materials, which contain the de-differentiated,programmable stem cells or target cells. In this regard, the carriermaterials can be bags or chambers for insertion into the human body.

[0104] In one embodiment of the invention such a bag, containing isletcells, which are differentiated somatic cells according to theinvention, is used for the production of a pharmaceutical construct foruse as an artificial islet cell port chamber for the supply of insulin.

[0105] According to a further embodiment of the invention, a bag orchamber containing adipocytes, which are differentiated somatic cellsaccording to the invention, is used for the production of an artificialpolymer filled with adipocytes as a pharmaceutical construct for breastconstruction after surgery and in the case of further indications ofplastic and/or cosmetic correction.

[0106] Moreover, semi-permeable port chamber systems, containingendocrine cells of very widely varying provenance, can be used in vivofor the treatment of endocrine, metabolic or hemostatic disorders.Examples of such endocrine cells are cells which produce thyroxine,steroids, ADH, aldosterone, melatonin, serotonin, adrenalin,noradrenalin, TSH, LH, FSH, leptin, cholecystokinin, gastrin, insulin,glucagon, or clotting factors.

[0107] Therefore, also implantable materials, which are semi-permeableport chamber systems, containing differentiated isolated somatic targetcells are subject of the invention. These semi-permeable chamber systemsare used in different embodiments of the invention for the production ofa pharmaceutical construct for the in-vivo treatment of endocrine,metabolic or hemostatic disorders.

[0108] The target cells obtained from the stem cells according to theinvention can in addition be used as cell cultures in bioreactorsoutside the body, for example in order to carry out detoxificationreactions. This form of use is particularly relevant in the case ofacute conditions, for example in the case of acute liver failure as ahepatocyte-bioreactor.

[0109] The production of the constructs illustrated above and conductingthe corresponding therapeutic process have already been illustrated manytimes in the state of the art, compare for example the review by Lalan,S., et al. “Tissue engineering and its potential impact on surgery”World J. Surg. 25: 1458-1466 (2001); Nasseri, B. A., et al. “Tissueengineering: an evolving 21st-century science to provide replacement forreconstruction and transplantation” Surgery 130: 781-784 (2001) andFuchs, J. R., et al., “Tissue engineering: a 21st century solution tosurgical reconstruction” Ann. Thorac. Surg. 72: 577-591(2001).

[0110] Finally, the pluripotent stem cells according to the inventionopen up a broad field for transgenic modification and therapy. Accordingto a preferred embodiment of the invention the dedifferentiatedprogrammable stem cells per se or somatic target cells and/or targettissue finally differentiated from these, are transfected with one ormore genes. In this way, one or more genes which are required tomaintain the metabolism of certain organs, such as for example livers orkidneys, are restored and/or supported or reintroduced. For example,stem cells or hepatocytes derived from these can be transfected with theFAH (fumaroylacetoacetate hydrolase) gene. In the FAH-deficient mousemodel the intrasplenic injection of 1000 FAH-positive donor hepatocyteswas sufficient to completely repopularize the liver after 6 to 8 weeksand fully compensate for the metabolic defect leading to cirrhosis ofthe liver (cf. Grompe, M., et al., Nat. Genet. 12: 266 ff. (1996)).

[0111] Correspondingly, by transfection of the stem cells or therespective target cells obtained from the stem cells by programming (forexample hematopoietic cells, hepatocytes, ovary cells, muscle cells,nerve cells, neurons, glia cells, cartilage or bones cells, etc.) with“Multi-Drug-Resistance-genes” extended radical chemotherapy can be madepossible in the case of malignant diseases by correspondinghematopoietic reconstitution or radiation resistance can be produced.

[0112] A starting material for the process according to the invention ismonocytes from human blood. These are preferably autologous monocytes,i.e., monocytes, which originate from the blood of the patient to betreated with the stem cells according to the invention or the targetcells produced from these.

[0113] To obtain the monocytes the blood can first, after standardtreatment with an anticoagulant in a known manner, preferably bycentrifugation, be separated into plasma and into white and red bloodcells. After the centrifugation the plasma is to be found in thesupernatant; below this lies a layer which contains the totality of thewhite blood cells. This layer is also referred to as “buffy coat”. Belowthis lies the phase containing red blood cells (hematocrit).

[0114] The “buffy coat” layer is then isolated and separated to obtainthe monocytes for example by centrifuging using a known process.According to a preferred process variant the “buffy coat” layer iscoated onto a lymphocyte separation medium (e.g., Ficoll Hypaque) andcentrifuged. By further centrifuging and rinsing, the monocyte fractionis obtained from the blood (cf. Example 1).

[0115] Examples of alternative processes for obtaining the monocytesfrom complete blood are “Fluorescence-Activated Cell Sorting” (FACS),“Immunomagnetic Bead Sorting” (cf. Romani et al., J. Immunol. Methods196: 137-151 (1996)) and “Magnetic-Activated Cell Sorting” (MACS) or theso called “Rosetting process” (cf. Gmelig-Meyling, F., et al.,“Simplified procedure for the separation of human T and non-T cells” VoxSang. 33: 5-8 (1977)).

[0116] According to the invention, monocytes can be obtained from anyisolated human blood, and the blood can also originate from organs suchas the spleen, lymph nodes or bone marrow. Obtaining monocytes fromorgans is considered especially when the separation of the monocytesfrom human blood, e.g., in the case of anemia or leukemia, is notpossible, or not in sufficient quantities, and in the case of allogenicuse, if, within the framework of multi-organ removal, the spleen isavailable as a source for isolation of monocytes.

[0117] For the production of a sufficient quantity of stem cellsaccording to the invention it is first necessary to propagate themonocytes. For this purpose, growth media suitable for monocytes can beused, wherein, according to the invention said medium contains M-CSF(macrophage colony stimulating factor). M-CSF (also referred to asCSF-1) is produced by monocytes, fibroblasts and endothelial cells. Theconcentration of M-CSF in the culture medium can amount to 2 to 20 μg/lmedium, preferably 4 to 6 μg/l and in a particularly preferred manner 5μg/l.

[0118] On the monocytes M-CSF binds to the specific c-Fms receptor (alsoreferred to as CSF-1R), which is exclusively present on the surface ofmonocytes and which only binds M-CSF (Sherr C. J., et al., Cell 41 (3):665-676 (1985)). As the specific interaction between M-CSF and thereceptor induces the division of the monocytes, the medium, in which themonocytes are cultivated contains M-CSF or an analogue thereof, whichcan bind to the receptor and activate it. Other growth factors such asGM-CSF (granulocyte-monocyte colony stimulating factor) and G-CSF(granulocyte colony stimulating factor) are unsuitable, as, due to thelack of affinity to the c-Fms receptor, they are not capable of inducingmonocyte division.

[0119] In a particularly preferred embodiment of the process M-CSF andIL-3 are simultaneously added to the cell culture medium in Step b) ofthe process. The concentration of IL-3 in the medium may amount to 0,2to 1 μg/l, preferably 0,3 to 0,5 μg/l and in a particularly preferredmanner 0,4 μg IL-3/l.

[0120] It is however also possible, to add initially only M-CSF to thecell culture medium in Step b) and add IL-3 only thereafter.

[0121] In a further embodiment the culture vessel initially containscell culture medium which contains only M-CSF, which after theseparation of the cells is then replaced by a second cell culturemedium, which contains IL-3.

[0122] According to a preferred embodiment of the invention the cells inStep b) of the process are additionally cultivated in the presence of asulfur compound, e.g., a mercapto compound, in which at least onehydrocarbon group is bonded to the sulfur, and said hydrocarbon group(s)may be substituted with one or more functional groups. Mercaptocompounds are defined as compounds which have at least one mercaptogroup (—SH), which is bonded to a hydrocarbon group. By the additionaluse of such a sulfur compound, the number of the stem cells obtained bydedifferentiation of the cells of monocytic origin, which express one ormore of the stem cell markers CD90, CD117, CD123 and CD135, can beincreased.

[0123] The functional group(s) is/are preferably hydroxyl- and/or aminegroups. In a particularly preferred embodiment, the sulfur compound is2-mercaptoethanol. According to a further preferred embodiment thesulfur compound is dimethylsulfoxide (DMSO).

[0124] The quantity of the sulfur compound used can range fromapproximately 4 to approximately 200 μmol/l relative to the sulfur.Approximately 100 μmol/l is preferred.

[0125] When 2-mercaptoethanol is used, the culture medium should containapproximately 3 μl to approximately 13 μl, preferably approximately 7 μl2-mercaptoethanol/l.

[0126] The treatment with IL-3 and optionally with the sulfur compoundcan be carried out simultaneously with or following the propagation ofthe monocytes by cultivation with M-CSF, simultaneous propagation andtreatment with IL-3 and optionally a sulfur compound being preferred.Propagation and dedifferentiation should, taken together, last no morethan 10 days, and the treatment with IL-3 and optionally with the sulfurcompound should be carried out over at least 3 and at most 10 days,preferably 6 days.

[0127] Therefore, according to the invention, in the case of cultivationof the monocytes in a culture medium, which simultaneously containsM-CSF, IL-3 and preferably a mercapto compound, the duration ofcultivation until the detaching of the cells from the bottom of theculture vessel amounts to at least 3 and at most 10 days, preferably 5to 8 days and particularly preferably 6 days.

[0128] If in a preferred embodiment the process according to theinvention is carried out in such a way that the monocytes in Step b) areinitially propagated in a medium containing only M-CSF, the propagationin such a culture medium can take place over a period of at least 2,preferably 3 and particularly preferably 4 days with a maximum durationof 7 days, and a subsequent cultivation in the presence of IL-3 andoptionally of a mercapto compound can take place over a further 3 days.Preferably in such a case the cultivation in a medium containing onlyM-CSF will however only last a maximum of 4 days, followed by acultivation in the presence of IL-3 and optionally of a mercaptocompound over a period of 3, 4, 5 or 6 days.

[0129] To carry out the propagation and dedifferentiation jointly, asillustrated in Examples 2 and 13, the monocytes are after isolationtransferred into a medium, which contains both M-CSF, and IL-3 as wellas preferably the sulfur compound, in particular mercaptoethanol orDMSO.

[0130] Due to their adhesive properties the monocytes and the stem cellsproduced from them during the process adhere to the bottom of therespective culture vessel. According to a preferred embodiment of theinvention, the culture medium is after Step c) separated from the cellsadhering to the bottom of the culture vessel and is discarded. This ispreferably followed by rinsing of the cells adhering to the bottom withculture medium, and the cells are then covered with fresh culture medium(cf. Example 13).

[0131] In this step the propagation and dedifferentiation mediumillustrated above can be used as culture medium, as well as a standardcell culture medium, for example RPMI.

[0132] According to a further preferred embodiment of the invention, thecells are brought into contact with a biologically well-toleratedorganic solvent at the end of Step c) and before Step d), in order toincrease the number of stem cells floating freely in the medium at theend of the process. The quantity of the solvent can range from 10 μl to1 ml. This is preferably an alcohol with 1-4 carbon atoms, the additionof ethanol being particularly preferred. According to a particularlypreferred embodiment the cells are brought into contact with the vaporphase of the previously defined biologically well-tolerated organicsolvent, preferably with ethanol vapor (cf. Example 2). The time forexposure to the organic solvent, particularly preferably to ethanolvapor, should amount to 4-12 hours, preferably 8-10 hours.

[0133] The process according to the invention is preferably carried outin culture vessels, the surface of which has previously been coated withfetal calf serum (FCS) (cf. Example 2). Alternatively human AB-Serumfrom male donors can be also be used. The coating with FCS can becarried out by covering the surface of culture vessels with FCS beforeuse, and after an exposure time of a few, in particular 2 to 12 hours,and in a particularly preferable manner 7 hours, and by removing the FCSnot adhering to the surface in a suitable manner.

[0134] If treatment with organic solvent take place after Step c)optionally after exchange of the culture medium, the cells alreadybecome detached from the bottom to a certain extent in this processstep. The (further) detaching can be carried out mechanically, forexample with a fine cell scraper, spatula or tip of a pipette (cf.Example 13).

[0135] According to a preferred embodiment of the process, completedetaching is carried out by treatment with a suitable enzyme, forexample with trypsin (cf. Example 2). The cells may be exposed to thetrypsin solution (0,1 to 0,025 g/l, preferably 0,05 g/l) for 2-10minutes at 35° C. to 39° C., preferably at 37° C., in the presence ofCO₂.

[0136] The trypsin activity is then blocked by a standard method, andthe now freely floating dedifferentiated programmable stem cells can beobtained by a standard method, for example by centrifuging and in oneembodiment by suspended in a suitable cell culture at the end of Stepd). They are now available, suspended in a suitable medium, for examplein RPMI 1640 or DMEM, for immediate differentiation into the desiredtarget cells. They can however also be stored in the medium for a fewdays. In a preferred embodiment the medium contains a cytokine or LIFfactor (leukemia inhibitory factor), cf. Nature 414: 94 (2001, Donovan,P. J., Gearhardt, J., loc. cit.), if the cells are to be stored inculture for longer than approximately 48 hours as dedifferentiatedprogrammable stem cells. In a medium containing such factors stem cellscan be kept for at least 10 days as dedifferentiated programmable stemcells.

[0137] In a preferred embodiment the cells are suspended for longerstorage in a liquid medium and then deep-frozen. Protocols for the deepfreezing of living cells are known in the state of the art, cf. GriffithM., et al. “Epithelial Cell Culture, Cornea, in Methods of TissueEngineering”, Atala A., Lanza R. P., Academic Press 2002, Chapter 4,Pages 131 to 140. A preferred suspension medium for the deep freezing ofthe stem cells according to the invention is FCS-containing DMEM, cf.Example 2.

[0138] The invention is further exemplified and illustrated below withreference to examples.

[0139] If not defined within the examples, the composition of the mediaand substances used are as follows:

[0140] 1. Penicillin/Streptomycin Solution:

[0141] 10,000 units of penicillin as sodium salt of penicillin G and1000 μg streptomycin as streptomycin sulfate per ml physiological sodiumchloride solution (NaCl 0,9%).

[0142] 2. Trypsin-EDTA

[0143] 0.5 g trypsin and 0.2 g EDTA (4 Na)/l

[0144] 3. Insulin

[0145] human, recombinant, produced in E. coli, approximately 28units/mg

[0146] 4. RPMI 1640 (1×, Liquid (11875)) Contains L-Glutamine

[0147] RPMI (Roswell Park Memorial Institute) Media 1640 are enrichedformulations, which can be used extensively for mammalian cells.Components Mol.-weight Conc. (mg/l) Molarity (nM) Anorganic saltsCalcium nitrate 236 100.00 0.424 (Ca(NO₃)₂ 4H₂O) Potassium chloride(KCl)  75 400.00 5.30 Magnesium sulfate 120 48.84 0.407 (MgSO₄) Sodiumchloride (NaCl)  58 6000.00 103.44 Sodium bicarbonate  84 2000.00 23.800(NaHCO₃) Sodium phosphate 142 800.00 5.63 (Na₂HPO₄) Further componentsGlucose 180 2000.00 11.10 Glutathione, reduced 307 1.50 0.0032 Phenolred 398 5.00 0.0125 Amino acids L-Arginine 174 200.00 1.10 L-Asparagine132 50.00 0.379 L-Asparaginic acid 133 20.00 0.150 L-Cysteinedihydrochloride 313 65.00 0.206 L-Glutaminic acid 147 20.00 0.136L-Glutamine 146 300.00 2.05 Glycine  75 10.00 0.133 L-Histidine 15515.00 0.0967 L-Hydroxyproline 131 20.00 0.153 L-Isoleucine 131 50.000.382 L-Leucine 131 50.00 0.382 L-Lysine hydrochloride 146 40.00 0.219L-Methionine 149 15.00 0.101 L-Phenylalanine 165 15.00 0.0909 L-Proline115 20.00 0.174 L-Serine 105 30.00 0.286 L-Threonine 119 20.00 0.168L-Tryptophan 204 5.00 0.0245 L-Tyrosine disodium, 261 29.00 0.110dihydrate L-Valine 117 20.00 0.171 Vitamins Biotin 244 0.20 0.008D-calcium pantothenate 477 0.25 0.0005 Choline chloride 140 3.00 0.0214Folic acid 441 1.00 0.0022 i-Inositol 180 35.00 0.194 Niacinamide 1221.00 0.0081 p-aminobenzoic acid 137 1.00 0.0072 (PABA) Pyridoxine HCl206 1.00 0.0048 Riboflavin 376 0.20 0.0005 Thiamin HCl 337 1.00 0.0029Vitamin B12 1355  0.005 0.00000369

[0148] Reference: Moore G. E., et al., J.A.M.A. 199: 519 (1967)

[0149] 5. PBS (Dulbecco's Phosphate Buffered Saline) cf. J. Exp. Med.98:167 (1954): Components g/l KCl 0.2 KH₂PO₄ 0.2 NaCl  8.00 Na₂PHO₄ 1.15

[0150] 6. 2-Mercaptoethanol

[0151] Quality for synthesis; Content>98%, Density 1.115 to 1.116, cf.e.g., Momo J., et al., J. Am. Chem. Soc. 73: 4961 (1951).

[0152] 7. Ficoll-Hypaque:

[0153] Lymphocyte separation medium(saccharose/epichlorohydrin-copolymerizate Mg 400,000; Density 1.077,adjusted with Sodium diatrizoate).

[0154] 8. Retinic Acid:

[0155] Vitamin A acid (C₂₀H₂₈O₂), 300 μl in 1.5 ml PBS corresponding to1 mM. As medium for programming of neurons and glia cells use 150 μl on10 ml medium (corresponding to 10⁻⁶ M).

[0156] 9. DMEM

[0157] Dulbecco's modified Eagle medium (high glucose) cf. Dulbecco, R.et al., Virology 8: 396 (1959); Smith, J. D. et al., Virology 12: 158(1960); Tissue Culture Standards Committee, In Vitro 6: 2 (1993)

[0158] 10. L-Glutamine

[0159] Liquid: 29.2 mg/ml

[0160] 11. Collagenase Type II:

[0161] Cf. Rodbell, M. et al., J. Biol. Chem. 239: 375 (1964).

[0162] 12. Interleukin-3 (IL-3):

[0163] Recombinant human L-3 from E. coli (Yang Y. C. et al., Cell 47:10 (1986)); contains the 133 amino acid residues including mature IL-3and the 134 amino acid residues including the methionyl form in a ratioof approximately 1:2; calculated mol. mass approximately 17.5 kD;specific activity 1×10³ U/μg; (R&D Catalogue No. 203-IL)

[0164] 13. Macrophage-Colony Stimulating Factor (M-CSF)

[0165] Recombinant human M-CSF from E. coli; contains as monomer (18.5kD) 135 amino acid residues including the N-terminal methionine; ispresent as a homodimer with a molar mass of 37 kD; (SIGMA Catalogue No.M 6518)

[0166] 14. Antibodies:

[0167] The antibodies used in the examples against the antigens CD14,CD31, CD90, CD117, CD123, CD135 are commercially available. They wereobtained from the following sources:

[0168] CD14: DAKO, Monoclonal Mouse Anti-Human CD14, Monocyte, CloneTÜK4, Code No. M 0825, Lot 036 Edition 02.02.01;

[0169] CD31: PharMingen International, Monoclonal Mouse Anti-Rat CD31(PECAM-1), Clone TLD-3A12, Catalogue No. 22711D, 0.5 mg;

[0170] CD90: Biozol Diagnostica, Serotec, Mouse Anti-Human CDw90, CloneNo. F15-42-1, MCAP90, Batch No. 0699;

[0171] CD117: DAKO, Monoclonal Mouse Anti-Human CD117, c-kit, Clone No.104D2, Code No. M 7140, Lot 016, Edition 04.05.00;

[0172] CD123: Research Diagnostics Inc., Mouse Anti-human CD123antibodies, Clone 9F5, Catalogue No. RDI-CD123-9F5;

[0173] CD135: Serotec, Mouse Anti-Human CD135, MCA1843, Clone No.BV10A4H2.

EXAMPLE 1

[0174] Separation of Monocytes from Whole Blood

[0175] To avoid blood clotting and to feed the cells, 450 ml of wholeblood in a 3-chamber bag set was mixed with 63 ml of a stabilizingsolution, which contained for each liter of H₂O, 3.27 g citric acid,26.3 g trisodium citrate, 25.5 g dextrose and 22.22 g sodiumdihydroxyphosphate. The pH-value of the solution amounted to 5.6-5.8.

[0176] “Sharp centrifugation” of this mixture was then carried out toseparate the blood components at 4000 rpm for 7 minutes at 20° C. Thisresulted in a 3-fold stratification of the corpuscular andnon-corpuscular components. By inserting the set of bags into a pressingmachine provided for this purpose, the erythrocytes were then pressedinto the lower bag, the plasma was pressed into the upper bag, and the“Buffy-coat” remained in the middle bag, and it contained approximately50 ml in volume.

[0177] The quantity of 50 ml freshly obtained “Buffy-coat” was thendivided into 2 portions of 25 ml each, each of which was then coatedwith 25 ml Ficoll-Hypaque separation medium, which had been introducedinto two 50 ml Falcon tubes beforehand.

[0178] This mixture was centrifuged without brake for 30 minutes at 2500rpm. Thereafter, erythrocytes and dead cells still present in the “Buffycoat” lay below the Ficoll phase while the white blood cells includingthe monocytes are separated as a white interphase on the Ficoll.

[0179] The white interphase of the monocytes was then carefully pipettedoff and was mixed with 10 ml of phosphate buffered physiological saline(PBS).

[0180] This mixture was then centrifuged with brake three times for 10minutes at 1800 rpm; the supernatant was pipetted off after eachcentrifugation and fresh PBS was filled up.

[0181] The cell sediment collected on the base of the centrifugationvessel (Falcon tube) contained the mononuclear cell fraction, i.e., themonocytes.

EXAMPLE 2

[0182] Propagation and Dedifferentiation of the Monocytes

[0183] The cultivation and propagation of the monocytes on the one handand the dedifferentiation of the cells on the other hand were carriedout in one step in nutrient medium of the following composition: RPMI1640 medium 440 ml Fetal calf serum (FCS)  50 ml Penicillin/Streptomycinsolution  5 ml 2-Mercaptoethanol (Stock solution)  5 ml Total volume 500ml

[0184] The nutrient medium further contained 2,5 μg/500 ml of M-CSF and0,2 μg/500 ml interleukin-3 (IL-3).

[0185] The monocytes isolated in Example 1 were transferred into 5chambers of a 6-chamber well plate (30 mm diameter per well) in aquantity of approximately 10⁵ cells per chamber in each case, and filledup in each case with 2 ml of the above-mentioned nutrient medium. The6-well plate was previously filled with pure, inactivated FCS and theFCS was decanted after approximately 7 hours, in order to obtain anFCS-coated plate in this way. The cell number for the exact dose perwell was determined according to a known process, cf. Hay R. J., “CellQuantification and Characterization” in Methods of Tissue Engineering,Academic Press 2002, Chapter 4, Pages 55-84.

[0186] The 6-well plate was covered with its lid and stored for 6 daysin an incubator at 37° C. The cells settled to the bottom of thechambers after 24 hours. Every second day the supernatant was pipettedoff and the chambers of the 6-well plate were again each filled up with2 ml of fresh nutrient medium.

[0187] On the 6th day 2 ml of 70% ethanol was introduced into the 6-wellplate's 6th chamber which had remained free, the plate was again closedand was stored for a further 10 hours at 37° C. in the incubator.

[0188] Subsequently, 1 ml of a trypsin solution diluted 1:10 with PBSwere pipetted into each of the chambers of the well plate whichcontained cells. The closed well plate was placed for 5 minutes at 37°C. under 5% CO₂ in the incubator.

[0189] The trypsin activity was subsequently blocked by the addition of2 ml of RPMI 1640 medium to each of the wells. The total supernatant ineach of the chambers (1 ml trypsin+2 ml medium) was pipetted off, pooledin a 15 ml Falcon tube and centrifuged for 10 minutes at 1800 rpm. Thesupernatant was then discarded and the precipitate was mixed with freshRPMI 1640 medium (2 ml/10⁵ cells).

[0190] This cell suspension could be directly used for differentiationinto different target cells.

[0191] Alternatively, after centrifugation and discarding of thetrypsin-containing supernatant the cells were mixed with DMSO/FCS as afreezing medium and deep-frozen at a concentration of 10⁶/ml.

[0192] The freezing medium contained 95% FCS and 5% DMSO. In each caseapproximately 10⁶ cells were taken up in 1 ml of the medium and cooleddown in the following steps:

[0193] 30 minutes on ice;

[0194] 2 hours at −20° C. in pre-cooled Styropor boxes;

[0195] 24 hours at −80° C. in Styropor;

[0196] Storage in tubes in liquid nitrogen (N₂) at −180° C.

[0197] For immune-histochemical phenotyping of the cell population ofdedifferentiated programmable stem cells of monocytic origin, generatedaccording to the above process, in each case 10⁵ cells were taken andfixed as a cytospin preparation on slides for further histochemicalstaining (Watson, P. “A slide centrifuge; an apparatus for concentratingcells in suspension on a microscope slide.” J. Lab. Clin. Med., 68:494-501 (1966)). After this the cells could be stained using thetechnique illustrated by Cordell, J. L., et al., (Literature, see below)with APAAP red complex. If not indicated otherwise, the added primaryantibody was diluted 1:100 with PBS, and in each case 200 μl of thisconcentration of antibodies was used. Monoclonal antibodies were used asprimary antibodies against the cell antigen epitopes listed in Table 1.FIG. 6 shows stained cytospin preparations and the corresponding proofof the stem cell markers CD90, CD117, CD123 and CD135.

[0198] Literature Relating to Staining Technique:

[0199] Cordell J. L., et al. “Immunoenzymatic labeling of monoclonalantibodies using immune complexes of alkaline phosphatase and monoclonalanti-alkaline phosphatase (APAAP complexes).” J. Histochem. Cytochem.32: 219-229 (1984).

[0200] Literature Relating to the Markers:

[0201] CD14

[0202] Ferrero E., Goyert S. M. “Nucleotide sequence of the geneencoding the monocyte differentiation antigen, CD14”, Nucleic Acids Res.16: 4173-4173 (1988).

[0203] CD31

[0204] Newman P. J., Berndt M. C., Gorski J., White J. C. II, Lyman S.,Paddock C., Muller W. A. “PECAM-1 (CD31) cloning and relation toadhesion molecules of the immunoglobulin gene superfamily”, Science 247:1219-1222 (1990).

[0205] CD90

[0206] Seki T., Spurr N., Obata F., Goyert S., Goodfellow P., Silver J.“The human thy-1 gene: structure and chromosomal location”, Proc. Natl.Acad. Sci. USA 82: 6657-6661 (1985).

[0207] CD117

[0208] Yarden Y., Kuang W. -J., Yang-Feng T., Coussels L., Munemitsu S.,Dull T. J., Chen E., Schlessinger J., Francke U., Ullrich A. “Humanproto-oncogene c-kit: a new cell surface receptor tyrosine kinase for anunidentified ligand.” EMBO J. 6: 3341-3351 (1987).

[0209] CD123

[0210] Kitamura T., Sato N., Arai K., Miyajima A. “expression cloning ofthe human IL-3 receptor cDNA reveals a shared beta subunit for the humanIL-3 and GM-CSF receptors.” Cell 66: 165-1174 (1991).

[0211] CD135

[0212] Small D., Levenstein M., Kim E., Carow C., Amn S., Rockwell P.,Witte L., Burrow C., Ratajazak M. Z., Gewirtz A. M., Civin C. I.,“STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed inCD34+ human bone marrow cells and is involved in the proliferation ofearly progenitor/stem cells.” Proc. Natl. Acad. Sci. USA 91: 459-463(1994). TABLE 1 Antigen expression of the stem cells according to theinvention Antigen Color reaction Stem cell marker CD90 ++ CD117 + CD123++ CD135 +(+) Differentiation marker CD14 (monocytes) +

[0213] The graduation indicated corresponds to the detected antigenpositivity, which becomes apparent from Day 4 to Day 9 after cultivationof the monocytes in the correspondingly specified media and was carriedout via microscopic comparison of the respective cytospin colorationswith the negative control (coloration observed without primaryantibodies).

[0214] Only cytospin preparations which had more than 70% vital cellswith typical stem cell morphology (cf. FIG. 6) were evaluated. Less than1% of these cells expressed the CD34 antigen.

EXAMPLE 3

[0215] Production of Neurons and Glia Cells from Adult Stem Cells

[0216] The production of neurons and glia cells was carried out in petridishes with a diameter of 100 mm. To prepare the petri dishes, 5 ml ofpure inactivated fetal calf serum (FCS) was introduced into each dish,so that the bottom was covered. After 7 hours, the proportion of FCS notadhering to the bottom of the petri dish was pipetted off. Approximately10⁶ of the cells produced in accordance with Example 2 were introducedinto one of the prepared petri dishes and 10 ml of nutrient medium ofthe following composition was added: DMEM solution 440 ml Fetal calfserum (FCS)  50 ml 1-Glutamine  5 ml Penicillin (100 U/l)/Streptomycin(100 μg/l) solution  5 ml Total volume 500 ml

[0217] The nutrient medium further contained retinic acid in a quantityof 1×10⁻⁶ M/500 ml.

[0218] The reprogramming/differentiation of the stem cells used intoneurons and glia cells took place within 10 days, the medium beingchanged at intervals of approximately 3 days. After this period, thecells were mostly adhering to the bottom of the chamber and could bedetached by brief trypsinization from the bottom of the plate in amanner analogous to that previously illustrated for the stem cells.

EXAMPLE 4

[0219] Evidence of Neuronal Precursor Cells, Neurons and Glia Cells

[0220] For the later immunohistochemical characterization of the targetcells induced by the dedifferentiated programmable stem cells, the stemcells generated from monocytes (10⁵ cells/glass lid) were applied toglass lids (20 mm×20 mm), which were placed on the bottom of the 6-wellplates (30 mm diameter per chamber) and cultivated with the nutrientmedium (2 ml) per well plate. After the respective target cells weredifferentiated, these were fixed as follows: After removal of thenutrient medium (supernatant) the cultivated target cells were fixed bythe addition of 2 ml Methanol, which took effect over 10 minutes.Subsequently the ethanol was pipetted off, and the well plates werewashed twice with PBS (2 ml in each case). After this, the cells couldbe stained with APAAP red complex using the technique illustrated byCordell, J. L., et al., “Immunoenzymatic labeling monoclonal antibodiesusing immune complexes of alkaline phosphatase and monoclonalanti-alkaline phosphatase (APAAP complexes).” J. Histochem. Cytochem.32: 219-229 (1994). Unless otherwise specified, the added primaryantibody was diluted 1:100 with PBS, in each case 200 μl of thisconcentration of antibodies were pipetted into each of the 6 wells.

[0221] Neuronal precursor cells were detected by staining the cells withthe antibody against the S100-antigen, cf. middle picture of FIG. 1(×200).

[0222] Neurons were detected by specific expression of synaptophysinMAP2 (microtubular associated protein 2) or neurofilament 68 with thecorresponding specific antibodies (primary antibody diluted 1:300 withPBS), right-hand picture of FIG. 1, ×200.

[0223] Glia cells, such as for example astrocytes, were identified bydetection of GFAP (glial fibrillary associated protein) (primaryantibody diluted 1:200 with PBS), left-hand picture of FIG. 1, ×200.

[0224] The separation of neurons and glia cells was carried out usingantibodies specific against MAP2 (neurons) or GFAP (glia cells), bymeans of MACS (Magnetic Activated Cell Sorting) according to the processas illustrated for example in Carmiol S., “Cell Isolation and Selection”Methods of Tissue Engineering, Academic Press 2002, Chapter 2, Pages19-35.

[0225] The cell types made visible by staining are shown in FIG. 1.

EXAMPLE 5

[0226] Production of Endothelial Cells from DedifferentiatedProgrammable Adult Stem Cells of Monocytic Origin

[0227] For the cultivation of endothelial cells, Matrigel® (Beckton andDickinson, Heidelberg, Del.) was used as matrix. This matrix consists offibronectin, laminin and collagens I and IV.

[0228] The frozen matrix was slowly thawed at 4° C. in a refrigeratorover a period of 12 hours. During this period its state changed, i.e.,the originally solid matrix became spongy/liquid. In this state it wasintroduced into a 48-well plate (10 mm diameter per well) in such amanner, that the bottom of each of the wells was covered.

[0229] After application, the plate was kept for 30 minutes at roomtemperature, until the gel had solidified at the bottom as an adherentlayer.

[0230] Subsequently approximately 1×10² cells per well were incubated onMatrigel® with addition of the nutrient medium (as illustrated inExample 2).

[0231] After 4-5 days the first tubular cell strands appeared, whichdeveloped after 6-8 days into three-dimensional cell networks. On thecells, the endothelial markers CD31 and factor VIII could be identifiedwith the respective specific primary antibodies (200 μl, in each casediluted to 1:100 with PBS).

[0232] In an alternative process the liquefied matrix was applied to avessel-prosthesis, which was then coated with the dedifferentiatedprogrammable adult stem cells according to Example 2. Afterapproximately 6 days a lawn of endothelial cells could be identified,which coated the prosthesis in a circular manner.

[0233] The endothelial cells made visible by staining with correspondingendothelium-specific antibodies (see above) are shown in FIG. 2. In themiddle picture, the cells are shown after 5 days' incubation onMatrigel®. First tubular strands combine individual cell aggregates. Thedark-brown marked cells express CD31 antigen (×200 with yellow filter).After 8 days there is an increasing formation of three-dimensionalnetwork structures takes place (anti-CD31-antigen staining, ×200 withyellow filter). After 12 days the newly differentiated CD31⁺ cells,which had been cultivated on Matrigel®, form a vessel-likethree-dimensional tube with multi-layer wall structures, which isalready morphologically reminiscent of a vessel. It is recognized, thatnow almost all the cells express the CD31 antigen (CD31 coloration,×400, blue filter), right-hand picture.

EXAMPLE 6

[0234] Production of Fat Cells (Adipocytes)

[0235] A: For the programming/differentiation of the adult stem cellsaccording to Example 2 into fat cells, a conditioned medium was firstgenerated. For this purpose 20 g of an autologous fat tissue, i.e., fattissue from the same human donor, from the blood of whom the monocytesalso originated, was processed as follows:

[0236] At first, the fat tissue was crushed in a petri dish and thecrushed tissue pieces were passed through a sieve (diameter of holes 100μm).

[0237] The suspension thus obtained was then transferred into a petridish with a diameter of 100 mm and 10 ml DMEM-medium with a content of30 mg collagenase type II were added. The mixture was left forapproximately 60 minutes at room temperature (22° C.±2° C.) to allow thecollagenase to take effect on the fat cells.

[0238] Subsequently the mixture was transferred to 50-ml Falcon tubes,and the tubes were centrifuged for 10 minutes at 1800 rpm.

[0239] After centrifugation the supernatant was discarded and the cellpellet consisting of adipocytes and precursor cells was taken up in 8 mlof a medium of the following composition and incubated in petri dishes(diameter 100 mm) for 10 days at 37° C. in an incubator: DMEM solution444.5 ml Fetal calf serum (FCS)   50 ml Insulin solution  0.5 mlPenicillin (100 U/l)/Streptomycin (100 μg/l) solution    5 ml Totalvolume   500 ml

[0240] The insulin solution contained 18 mg insulin (Sigma 1-0259)dissolved in 2 ml of acetic water (consisting of 40 ml of H₂O and 0.4 mlof glacial acetic acid). The solution is diluted 1:10 with acetic water.

[0241] During the incubation over 10 days, the fat-cell-conditionedmedium (FCCM) formed a supernatant. The supernatant was replaced withfresh nutrient medium after 2 to 4 days in each case. The FCCM obtainedduring each change of medium was subjected to sterile filtration andstored at −20° C. Subsequently 10 ml of the FCCM illustrated above wereintroduced into a petri dish (diameter 100 mm) together withapproximately 10⁶ stem cells according to Example 2. The first precursorcells containing fat vacuoles became visible after 4 days (FIG. 3A).After 6 days, single adipocytes appeared, which could be stained withSudan red (FIGS. 3B and C). After 10 days there was typical aggregationand cluster formation of these cells, which at this step could alreadybe observed macroscopically as fat tissue (FIG. 3D).

[0242] The fat cells made visible by staining in FIGS. 3A-3D thus differquite considerably from the controls 3E and 3F: FIG. 3E shows the cellsof monocytic origin, which were cultivated in the nutrient medium (asindicated in Example 2) for 6 days, but without the addition of IL-3 and2-mercaptoethanol to the nutrient medium. This was followed by theaddition of the FCCM. These cells were not capable of differentiatinginto fat cells. FIG. F shows cells, which were cultivated for 6 dayswith complete medium (according to Example 2), and which were thentreated for a further 6 days with nutrient medium instead of with FCCM(according to Example 2). The FCCM thus contains components which arerequired to provide the signal for differentiation into fat cells.

[0243] The staining of the cells with Sudan red in FIGS. 3A, B, C and Dtook place according to the method illustrated by Patrick Jr., C. W., etal. “Epithelial Cell Culture: Breast”, in Methods of Tissue Engineering,Academic Press 2002, Chapter 4, Pages 141-149.

[0244] B: In addition to the phenotyping of the fat cells by stainingwith Sudan red, molecular-biological characterization of the fat cellswas carried out at the mRNA level, in order to check whether the geneticprogram of the fat cells, after corresponding programming with thefat-cell-conditioning medium used, undergoes a corresponding alteration,and typical messenger-ribonucleic acid (mRNA) transcripts, illustratedfor fat cells can be identified in the fat cells programmed fromprogrammable monocytes. Two mRNA sequences typical of fat cellmetabolism were amplified by means of polymerase chain reaction (PCR)from isolated RNA samples from dedifferentiated programmable stem cellsof monocytic origin and, in a parallel test mixture, amplified from theprogrammed fat cells, namely “peroxisome proliferative activatedreceptor gamma” (PPARG)-mRNA, (Tontonoz, P., et al. “Stimulation ofadipogenesis in fibroblasts by PPAR gamma 2, a lipid-activatedtranscription factor.” Cell 79: 1147-1156 (1994), gene bank access codenumber; NM_(—)005037) and “leptin (obesity homolog, mouse)”-mRNA, (ZhangY., et al. “Positional cloning of the mouse obese gene and its humanhomologue.” Nature 372: 425-432 (1994), gene bank, access code number:NM_(—)000320).

[0245] The RNA-isolation needed for this purpose, the reversetranscription method and the conditions of the PCR amplification of thedesired mRNA sequences were carried out as illustrated in detail in thestate of the art, see Ungefroren H., et al., “Human pancreaticadenocarcinomas express Fas and Fas ligand yet are resistant toFas-mediated apoptosis”, Cancer Res. 58: 1741-1749 (1998).

[0246] For this purpose the respective primers produced for the PCRamplification were selected so that the forward- and reverse primersbind to mRNA sequences, whose homologous regions in the chromosomal genelie in two different exons and are separated from one another by a largeintron. It could thereby be ensured that the amplification fragmentobtained originates from the mRNA contained in the cell and not from thesequence present in the chromosomal DNA. In particular the followingprimer sequences were selected for PPAR-γ and for leptin:

[0247] PPAR-γ: forward-primer; 265-288 (corresponding gene sequence inexon 1), reverse-primer: 487-465 (corresponding gene sequence in exon2), this results in an amplification-fragment of 487-265 bp=223 bp, seeFIG. 3G. As further shown by FIG. 3G traces of transcribedPPAR-γ-specific mRNA can already be identified in the programmable stemcell and in the tumor cell line HL-60 (of a human promyeloic leukemiacell line), although with significantly narrower signal bands than inthe fat cell itself. In contrast, the fat-cell-specific protein leptincan only be detected in the fat cells derived from the programmable stemcells at mRNA level by reverse-transcriptase PCR.

[0248] The programmable stem cells (progr. stem cell) used as a controland the human tumor cell lines HL-60, Panc-1 and WI-38 transcribe noleptin. As negative controls all the samples without the addition of thereverse transcriptase (fat cel/-RT) and H₂O-samples were simultaneouslyco-determined. By identification of the GAPDH “house-keeping” gene inthe positive controls, it is ensured that the respective PCRamplification steps were properly carried out in the individualmixtures.

EXAMPLE 7

[0249] Production of Liver Cells (Hepatocytes)

[0250] A: For the programming of the dedifferentiated programmable stemcells of monocytic origin according to Example 2 into liver cells, aconditioned medium was first generated. For this purpose 40 g of humanliver tissue was processed as follows.

[0251] First the liver tissue was rinsed several times in PBS, toessentially remove erythrocytes. The tissue was then crushed in a petridish and incubated with a dissociation solution for approximately 45minutes at room temperature. The dissociation solution consisted of 40ml PBS (phosphate buffered saline), 10 ml of a trypsin solution diluted1:10 with PBS and 30 mg collagenase type II (Rodbel M., et al. J. Biol.Chem. 239: 375 (1964)). After 45-minutes' incubation the tissue pieceswere passed through a sieve (see Example 6).

[0252] The mixture was then transferred into 50-ml Falcon tubes, filledup to 50 ml with PBS and centrifuged for 10 minutes at 1800 rpm.

[0253] After centrifugation the supernatant was discarded and the cellpellet containing the liver cells was again washed with 50 ml PBS andcentrifuged. The supernatant thus produced was again discarded and thecell pellet taken up in 25 ml of a medium of the following compositionand incubated in cell culture flasks (250 ml volume) for 10 days at 37°C. in an incubator: Liver cell growth medium Liver cell growth medium,LCGM RPMI 1640 medium 445 ml Fetal calf serum (FCS)  50 ml Insulinsolution  0.5 ml  Penicillin (100 U/l)/Streptomycin (100 μg/l) solution 5 ml Total volume 500 ml

[0254] The nutrient medium contained in addition 5 μg (10 ng/ml) ofepidermal growth factor (Pascall, I. C. et al., J. Mol. Endocrinol. 12:313 (1994)). The composition of the Insulin solution was as illustratedin Example 6.

[0255] During the incubation lasting 10 days the liver cell conditionedmedium (LCCM) formed as a supernatant. The supernatant was replaced byfresh nutrient medium after 2 to 4 days respectively. The respectiveLCCM obtained during the change of medium in each case was subjected tosterile filtration (filter with 0.2 μm pore size) and stored at −20° C.

[0256] 1×10⁶ dedifferentiated stem cells were then cultivated with 10 mlof a medium of the following composition in a petri dish (Ø100 mm) or aculture flask. Liver cell differentiation medium (Liver celldifferentiation medium, LCDM): LCCM 100 ml Insulin solution (cf. Example6) 0.1 ml epidermal growth factor 1 μg hepatocyte growth factor 2 μg

[0257] Hepatocyte growth factor (Kobayashi, Y. et al., Biochem. Biophys.Res. Commun. 220: 7 (1996)) was used in the concentration of 40 ng/ml.After a few days morphological changes towards flat, polygonal mono- ordiploid cells could be observed (FIG. 4A). After 10-12 days hepatocytesarising from dedifferentiated stem cells could be identified byimmune-histochemical detection of the liver-specific antigenalpha-fetoprotein (Jacobsen, G. K. et al., Am. J. Surg. Pathol. 5:257-66 (1981)), as shown in FIGS. 4B and 4C.

[0258] B: In addition to the phenotyping of the hepatocytes byimmune-histochemical identification of the alpha-fetoprotein, amolecular-biological characterization of the hepatocytes at mRNA levelwas carried out, in order to check whether the genetic program of thestem cells, after corresponding programming with theliver-cell-conditioning medium used undergoes a correspondingalteration, and whether messenger-ribonucleic acid (mRNA) transcripts,illustrated as typical of liver cells in the hepatocytes arising fromthe stem cells according to the invention can be identified. For thispurpose, the presence of five different mRNA sequences typical ofhepatocytes was examined by means of polymerase chain reaction (PCR) inisolated RNA samples from dedifferentiated programmable stem cells ofmonocytic origin and, in a parallel test sample, from the liver cellsobtained by programming of the stem cells. In particular, this is theHomo sapiens albumin-mRNA (Lawn, R. M., et al. “The sequence of humanserum albumin cDNA and its expression in E. coli.” Nucleic Acids Res. 9:6103-6114, (1981), gene bank access code number: NM-000477),alpha-fetoprotein-mRNA (Morinaga T., et al. “Primary structures of humanalpha-fetoprotein and its mRNA.” Proc. Natl. Acad. Sci. USA 80:4604-4608 (1983), gene bank access code number: V01514), Human carbamylphosphate synthetase I mRNA (Haraguchi, Y., et al. “Cloning and sequenceof a cDNA encoding human carbamyl phosphate synthetase I: molecularanalysis of hyper-ammonemia” Gene 107: 335-340 (1991), gene bank accesscode number D90282), Homo sapiens coagulation factor II (Thrombin, F2)mRNA (Degen, S. J. et al. “Characterization of the complementarydeoxyribonucleic acid and gene coding for human prothrombin”Biochemistry 22: 2087-2097 (1983), gene bank access code numberNM-000506), Homo sapiens coagulation factor VII (serum prothrombinconversion accelerator, F7) mRNA (NCBI Annotation Project. DirectSubmission, Feb. 6, 2002, National Center for Biotechnology Information,NIH, Bethesda, Md. 20894, USA, gene bank access code number XM-027508).

[0259] The RNA-isolation necessary for this reverse transcriptase methodand the conditions of the PCR amplification of the desired mRNAsequences was carried out as illustrated in detail in the state of theart, see Ungefroren H., et al., “Human pancreatic adenocarcinomasexpress Fas and Fas ligand yet are resistant to Fas-mediated apoptosis”Cancer Res. 58: 1741-1749 (1998).

[0260] The respective primers for the PCR amplification were selected sothat the forward- and reverse primers bind to mRNA sequences whosehomologous regions in the chromosomal gene lie in two different exonsand are separated from one another by a large intron. In this way itcould be ensured that the amplification fragment obtained originatesfrom the mRNA contained in the cell and not from the sequence present inthe chromosomal DNA.

[0261] The primer sequences indicated below were selected; the resultsof the respective PCR analyses are reproduced in FIG. 4D. Thededifferentiated programmable stem cells according to the invention, aredesignated there as “progr. stem cell” and the hepatocytes derived byprogramming of these as “progr. hepatocyte”.

[0262] Alpha-fetoprotein: forward primer: 1458-1478 (corresponding genesequence in Exon 1), reverse primer: 1758-1735 (corresponding genesequence in Exon 2), this results in an amplification fragment of1758-1458 bp=391 bp, see FIG. 4D.

[0263] As shown in FIG. 4, the programmable stem cell (progr. stemcell), which itself contains no identifiable specific mRNA transcriptsfor alpha-fetoprotein, can be programmed into a hepatocyte (progr.hepatocyte), which contains this mRNA transcript (positive band with amolecular weight of 301 bp). This also explains the immunohistochemicaldetectability of the alpha-fetoprotein, as shown in FIGS. 4B and 4C. Thepositive controls, namely human liver tissue and the liver tumor cellline HepG2 also transcribe alpha-fetoprotein-specific mRNA, as the 301bp bands confirm.

[0264] Albumin: forward primer: 1450-1473 (corresponding gene sequencein exon 1), reverse primer: 1868-1844 (corresponding gene sequence inExon 2), this resulted in an amplification fragment of 1868-1450 bp=419bp, see FIG. 4D.

[0265]FIG. 4D shows traces of transcribed albumin-specific mRNA alreadyin the programmable stem cell, while the hepatocytes obtained byprogramming of the stem cells and normal liver tissue as well as thetumor cell line HepG2, which were both used as positive controls,strongly express the mRNA, as can be recognized by clear bands.

[0266] The carbamyl phosphatase synthetase I: forward primer: 3135-3157(corresponding gene sequence in exon 1), reverse primer: 4635-4613(corresponding gene sequence in exon 2), this results in anamplification fragment of 4635-3135=1500 bp, see FIG. 4D.

[0267] The carbamyl phosphate synthetase I represents an enzyme specificto the hepatocytes, which plays an important role in the metabolizationof urea in the “urea cycle”. This detoxification function is guaranteedby functioning hepatocytes. As FIG. 4D shows, both in the hepatocytesgenerated from programmable stem cells and also in the positive controls(human liver tissue and the HepG2-tumor cell line), the mRNA bands (1500bp) specific to carbamyl phosphate synthetase I can be identified. Thesomewhat weaker expression of the mRNA bands for the programmedhepatocytes (progr. hepatocyte) is due to the lack of substrateavailable in the culture dish.

[0268] Clotting factor II: forward primer: 1458-1481 (corresponding genesequence in exon 1), reverse primer: 1901-1877 (corresponding genesequence in exon 2), this results in an amplification fragment of1901-1458=444 bp, see FIG. 4D.

[0269] This likewise hepatocyte-specific protein can only be detected inthe programmed hepatocyte (progr. hepatocyte) and in the positivecontrol from human liver tissue at mRNA level by 444 bp band expression,whereas the programmable stem cell (progr. stem cell) does not show thisband, i.e., the gene is not transcribed there, as can be seen in FIG.4D.

[0270] Clotting factor VII: forward primer: 725-747 (corresponding genesequence in exon 1), reverse primer: 1289-1268 (corresponding genesequence in exon 2), this results in an amplification fragment of1289-725=565 bp, see FIG. 4D.

[0271] As in the case of clotting factor II, also this protein is onlytranscribed in programmed hepatocytes (progr. hepatocyte) and in thepositive control (human liver tissue) (see bands at 656 bp), althoughweaker than clotting factor II. Neither the programmable stem cell northe negative control (H₂O) show this specific mRNA band.

[0272] Glycerine aldehyde dehydrogenase: This gene, also referred to asa “house-keeping gene” can be detected in every eukaryotic cell andserves as a control whether PCR amplification was properly carried outin all samples; it is co-determined in parallel and results from theaddition of a definite quantity of RNA from the respective cell samples.

[0273] As negative control H₂O samples were simultaneously co-determinedin all tests. If the H₂O is not contaminated with RNA, no amplificate isproduced during the PCR and no band is detectable (thus serves ascounter-control).

EXAMPLE 8

[0274] Production of Skin Cells (Keratinocytes)

[0275] For the programming of dedifferentiated programmable stem cellsof monocytic origin according to Example 2 in skin cells a conditionedmedium was first generated. For this purpose, 1-2 cm² of complete humanskin was processed as follows.

[0276] The skin material was first freed from the subcutis under sterileconditions. The tissue was then washed in total 10× with PBS in asterile container by vigorous shaking. After the 2nd washing, the tissuewas again freed from demarked connective tissue residues.

[0277] The skin material was then placed in a petri dish with a diameterof 60 mm, mixed with 3 ml of a trypsin solution diluted 1:10 with PBSand cut into small pieces (approximately 0.5 to 1 mm³). After this, 3 mlof the trypsin solution diluted 1:100 with PBS was again added to themixture and the mixture was incubated at 37° C. for 60 minutes withintermittent shaking.

[0278] The larger particles were then allowed to settle and thesupernatant containing the keratinocytes was poured off and centrifugedat 800 rpm for 5 minutes. The supernatant now produced was pipetted offand the cell pellet was taken up in 3 ml of a medium of the followingcomposition and incubated in petri dishes (Ø100 mm) for 15 days in anincubator at 37° C. Keratinocyte growth medium (Keratinocyte growthmedium, KGM): DMEM 333.5 ml Fetal Calf serum (FCS)   50 ml Ham's F12medium   111 ml Penicillin (100 U/l)/Streptomycin (100 μg/l) solution   5 ml Insulin solution (cf. Example 6)  0.5 ml Total volume   500 ml

[0279] The nutrient medium contained 5 μg of epidermal growth factor(for exact specification see Example 7) and 5 mg of hydrocortisone (Ref.Merck Index: 12, 4828).

[0280] During the 15 days' incubation period, thekeratinocyte-cell-conditioned medium KCCM formed as supernatant. Thesupernatant was replaced with fresh nutrient medium after 2-4 days ineach case. The KCCM obtained during each change of medium was subjectedto sterile filtration and stored at −20° C.

[0281] 1×10⁶dedifferentiated stem cells were then cultivated with 10 mlof a mediums of the following composition in a petri dish (Ø100 mm) or aculture flask. Keratinocyte differentiation medium (Keratinocytedifferentiation medium, KDM) KCCM 100 ml Insulin solution (cf. Example6) 0.5 ml epidermal growth factor (EGF) 1 μg Hydrocortisone 1 mgkeratinocyte growth factor (KGF) 2.5 μg

[0282] Keratinocyte growth factor was used in a concentration of 25ng/ml, as illustrated by Finch et al., Gastroenterology 110: 441 (1996).

[0283] After a few days a morphological change in the cells could beobserved. After 6 days the keratinocyte-specific antigens, cytokeratin 5and 6, which are both bound by the primary antibody used, (Exp. Cell.Res. 162: 114 (1986)) could be detected (FIG. 5A). After 10 days a celladherence of the clearly larger individual cells already took place inculture, which made it possible to identify a visible cell tissuecombination of confluent cells (FIG. 5B).

EXAMPLE 9

[0284] Production of Insulin-Producing Cells from DifferentiatedProgrammed Stem Cells

[0285] The production of insulin-producing cells was conducted inculture flasks with a volume of approximately 250 ml and flat walls (T75cell culture flasks). Approximately 5×10⁶ of the cells producedaccording to Example 13 were suspended in approximately 5 ml of theculture medium indicated below (differentiation medium for insulinproducing cells) and after being introduced into the flasks, mixed witha further 15 ml of culture medium. For the differentiation of the cells,the flasks were incubated in a horizontal position in an incubator at37° C. and 5% CO₂.

[0286] Culture medium (modified according to Rameya V. K. et al., NatureMedicine, 6 (3), 278-282 (2000)): RPMI 1640 445 ml Fetal calf serum(FCS) 50 ml Penicillin (100 U/l)/Streptomycin (100 μg/l) solution 5 mlNicotinamide 620 mg Glucose 360 mg Total volume 500 ml

[0287] The nutrient medium further contained the epidermal growth factorin a quantity of 10 ng/ml and the hepatocyte growth factor in a quantityof 20 ng/ml.

[0288] Within the first hour the cells adhere to the bottom of theculture vessel. The differentiation of the stem cells was monitored byreference to insulin production. For this purpose the culture medium waschanged at intervals of approximately 2 to 3 days, the cell supernatantwas collected each time, and frozen at −20° C. The cells adhering to thebottom of the culture flask could be detached by trypsinization asillustrated in Example 2.

[0289] The insulin content of the supernatant collected at the differenttimes was measured by means of ELISA (Enzyme-linked-immunosorbent-assay)against human insulin (Bruhn H. D., Fölsch U. R. (Eds.), Lehrbuch derLabormedizin: Grundlagen, Diagnostik, Klinik Pathobiochemie [Textbook ofLaboratory Medicine, Principles, Diagnosis, Clinical Pathobiochemistry](1999), Page 189) and compared with the blank reading of the medium. Theresults reproduced in FIG. 8 show that the cells have reached themaximum level of insulin production after 14 days in culture. Theinsulin quantities produced by the cells treated in the course of thedifferentiation increased after 14 days to 3 μU/ml, while no humaninsulin was detectable in the control medium. The bars in FIG. 8 eachrepresent three separate values each determined from three independentindividual experiments.

[0290] Next to the determination of the insulin production in thedeprogrammed stem cells, which were differentiated into insulinproducing cells according to the invention, the portion ofinsulin-producing cells were determined which still expressed themonocyte-specific surface antigen CD14 also 3 weeks after conducting thededifferentiation. It was found that on a great portion of these cells(about 30 to 40%) the monocyte-specific antigen CD14 was detectable alsoafter 3 weeks.

EXAMPLE 10

[0291] Alternative Method for the Production of Hepatocytes fromDedifferentiated Programmable Stem Cells

[0292] As an alternative to the use of hepatocyte-conditioned medium(LCCM), as illustrated in Example 7, the differentiation of the stemcells into hepatocytes was induced by the nutrient medium (Ha) indicatedbelow. The production of hepatocytes from stem cells in turn took placein culture flasks with a volume of approximately 250 ml and flat walls(T75-cell culture flasks). Approximately 5×10⁶ of the cells producedaccording to Example 13 were introduced into approximately 5 ml of theimproved culture medium indicated below (Ha, differentiation medium forhepatocytes) and after being introduced into the flasks, mixed with afurther 15 ml of culture medium. For the differentiation of the cells,the flasks were incubated in a horizontal position in an incubator at37° C. and 5% CO₂.

[0293] Differentiation medium for hepatocytes (Ha) (modified accordingto Schwarz et al., “Multi-potent adult progenitor cells from bone marrowdifferentiate into functional hepatocyte-like cells”, J. Clin. Invest.10 (109), 1291-1302 (2002)): RPMI 1640 445 ml Fetal calf serum (FCS)  50ml Penicillin (100 U/l)/Streptomycin (100 μg/l) solution  5 ml Totalvolume 500 ml

[0294] The nutrient medium also contained fibroblast growth factor-4(FGF-4) in a quantity of 3 ng/ml.

[0295] Within the first hour the cells adhere to the bottom of theculture vessel. The differentiation of the stem cells was monitored withregard to albumin production. For this purpose the culture medium waschanged at intervals of approximately 2 to 3 days, the cell supernatantcollected each time, and frozen at −20° C. The cells adhering to thebase of the culture flask could be detached by trypsinization asillustrated in Example 2.

[0296] The albumin content of the supernatant collected at the differenttimes was measured by means of ELISA (Enzyme-linked-immunosorbent-assay)for human albumin (according to the protocol of Bethyl Laboratories Inc.and according to Schwarz et al., loc. cit.) and compared with the blankreading of the medium. The results presented in FIG. 9 show that thealbumin production of the cells during the period of 14 to 28 days inculture remained approximately constant. The measurements were carriedout on days 0 (blank reading of the medium), 14, 21, 28 and 30 relativeto the time of addition of the Ha medium. The values determined in eachcase amounted to ca. 5 ng/ml, 450 ng/ml, 425 ng/ml, 440 ng/ml and 165ng/ml. The bars in FIG. 9 each represent three separate values eachdetermined from three independent individual experiments.

EXAMPLE 11

[0297] Determination of the Co-Expression of Albumin and of theMonocyte-Specific Antigen CD14 in Hepatocytes Derived fromDedifferentiated Stem Cells

[0298] The determination of the co-expression of albumin and of themonocyte-specific antigen CD14 in hepatocytes derived fromdedifferentiated stem cells was carried out on the one hand bydouble-staining (A) and on the other hand by FACS analysis (B).

[0299] A) Stem cells according to the invention differentiated intohepatocytes according to Example 10 were cultivated on cover glasses ina 6-well plate and fixed with methanol as illustrated in Example 4. Adouble-staining was then carried out, in order to detect thesimultaneous expression of the antigen CD14 (phenotype marker ofmonocytes) on the one hand and of albumin (liver-specific marker) on theother hand.

[0300] For this purpose the cells were first incubated as illustrated inExample 4 with a primary antibody against human albumin (guinea pig vs.human albumin) in a 1:50 dilution in PBS. Following a washing step, thecells were then incubated for 45 minutes with a secondary antibody mouseanti-rat, which binds the guinea pig antibodies, also in a 1:50 dilutionin PBS. The staining process was then carried out according to Example 4using the method of Cordell J. L., et al. (loc. cit.) with APAAP redcomplex.

[0301] For the second staining step, the cells were then incubated withthe primary antibody, mouse anti-human-CD14, and following a washingstep according to Example 4 stained with the ABC Streptavidin KIT ofVectastain (Vector) using the method of Hsu, S. M., et al. “The use ofantiavidin antibody and avidin-biotin-peroxidase complex inimmunoperoxidase technics” Am. J. Clin. Pathol. 75 (6): 816-821 (1981)with dem DAB-Complex (brown) (Vector Laboratories).

[0302] Nucleus counter-staining with haemalaun was then carried out asillustrated in Example 4, followed by embedding in Kaiser's glycerolgelatin.

[0303] The results are shown in FIG. 10. The figure shows the expressionof the antigen CD14 as brown color, which slowly decreases parallel tothe morphological transformation of the cells into hepatocytes, whilethe albumin expression as red color increases with the increasingmaturation of the hepatocytes. Picture No. 4 in FIG. 10 shows the cellsafter three weeks' stimulation with the hepatocyte-conditioned medium.

[0304] B) Parallel with the double marking, the stem cellsdifferentiated into hepatocytes according to the invention weresubjected to FACS (fluorescence-activated cell sorting) analysis.

[0305] The stem cells differentiated into hepatocytes according to theinvention according to Example 10 were first harvested by mechanicaldetachment of the cells from the culture flask using a cell scraper. Thecells were carefully rinsed from the flask with PBS and washed twice,each time in 10 ml of PBS-solution. For this purpose the cellsuspensions in the PBS solution were introduced into a 15-ml centrifugetube and precipitated at 1600 rpm. The resultant cell sediment wasdiluted with PBS, such that exactly 1×10⁵ cells were present in 100 μlPBS.

[0306] 10 μl of each of FITC-marked anti-CD14 antibodies (BD Pharmingen)or FITC-marked anti-albumin antibodies (Beckmann) and FITC-markednon-specific IgG1 mouse anti-human antibodies were then added to thiscell suspension. After an incubation period of 20 minutes the cells wereresuspended twice in 500 μl PBS and each precipitated for 5 minutes at1600 rpm and then finally taken up in 200 μl PBS. After resuspension ofthe cells, fluorescence was measured with a BD FACScalibur flowcytometer from the company BD Biosciences (Franklin Lakes, N.J.) (cf.Bruhn H. D., Fölsch U. R. (Eds.), Lehrbuch der Labormedizin: Grundlagen,Diagnostik, Klinik Pathobiochemie [Textbook of Laboratory Medicine,Principles, Diagnosis, Clinical Pathobiochemistry], 395-403 (1999); andHolzer U. et al., “Differential antigen sensitivity and costimulatoryrequirements in human Th1 and Th2 antigen-specific CD4(+) cells withsimilar TCR avidity” J. Immunol. 170 (3): 1218-1223 (2003)). Theevaluation of the results was carried out using the Microsoft WinMDIprogram with reference to Marquez M. G., et al. “Flow cytometricanalysis of intestinal intra-epithelial lymphocytes in a model ofimmunodeficiency in Wistar rats.” Cytometry 41 (2): 115-122 (2000).

[0307] The results of the FACS-Analysis are reproduced in FIG. 11. Thefigure shows the expression of the CD14 (top row) and of the albuminantigen (bottom row), which was measured in dedifferentiated monocytes(left-hand column) and in the stem cells differentiated into hepatocytesaccording to the invention (right-hand column). In dedifferentiatedmonocytes a strong expression of CD14, but no expression of albumincould be detected, while in the hepatocytes developed fromdedifferentiated monocytes a weaker expression of the CD14 and a verystrong expression of the albumin was detectable.

EXAMPLE 12

[0308] In Vivo Use of Dedifferentiated Programmed Stem Cells ofMonocytic Origin

[0309] In order to clarify, to what extent the programmable stem cellsin vivo after injection via the portal vein into the liver of agenetically identical recipient animal undergo a specificdifferentiation via the signal-providers present in the liver, livers offemale LEW rats were first treated with retrorsine, in order to inhibitthe hepatocytes present in the liver (liver parenchyma cells) regardingtheir proliferation activity (Ref. Lacone, E., et al. “Long-term,near-total liver replacement by transplantation of isolated hepatocytesin rats treated with retrorsine” Am. J. Path. 153: 319-329 (1998)).

[0310] For this purpose the LEW rats received 30 mg of the pyrrolizidinealkaloid retrorsine, injected intraperitoneally twice within 14 days.Subsequently an 80% resection of the livers treated in this way wascarried out, followed by the administration of 5×10⁵ of the programmablestem cells in 1 ml PBS into the portal vein of the remaining residualliver. The stem cells had been obtained, as illustrated in Example 2,from monocytes of male LEW rats. Five days after administration of thestem cells a punch biopsy of the liver was carried out for histologicalassessment of the liver and to detect the cell types differentiated fromthe stem cells by means of fluorescence-in-situ-hybridization (FISH)with Y-chromosome-specific probes, as illustrated in detail in Hoebee,B. et al. “Isolation of rat chromosome-specific paint probes bybivariate flow sorting followed by degenerate oligonucleotideprimed-PCR.” Cytogenet. Cell Genet. 66: 277-282 (1994).

[0311]FIG. 7A shows the Y-chromosome-positive (red points in the cellnucleus) hepatocytes derived from the male LEW stem cells on the 5th dayafter intraportal injection into retrorsine-pretreated 80%-resectionedlivers of female recipient animals. The selective removal of the sameliver on day 25 after stem cell injection shows the differentiation ofthe stem cells into hepatocytes, endothelial cells and bile ductepithelia (FIG. 7B). At this point in time, the liver has alreadyreached its normal size, and >90% of the cells have a Y-chromosome. Fromthis, it can be concluded, that the injected syngenic programmable stemcells of monocytic origin in are capable in vivo, of effecting acomplete restoration of the liver with normal metabolic function. FIG.7C shows in this connection the Kaplan-Meier survival curves (n=4 pergroup) of stem-cell-treated versus untreated recipient rats followingadministration of retrorsine and 80% liver resection.

[0312] The function parameters bilirubin and ammonia (NH₃) prove thecomplete metabolic functionality of the long-term survivingstem-cell-treated animals (FIGS. 7D and 7E).

EXAMPLE 13

[0313] Propagation and Dedifferentiation of Monocytes in Cell CultureFlasks

[0314] Cultivation and propagation of the monocytes on the one hand andthe dedifferentiation of the cells of the other side on a larger scalewere conducted in culture flasks in the same nutrient medium, which wasalso used for the cultivation in well-plates (cf. Example 2). Thenutrient medium contains 2.5 μg/500 ml M-CSF and 0.2 μg/500 mlinterleukine 3 (IL-3).

[0315] The monocytes isolated in Example 1 were transferred to thebottom of culture flasks having a volume of 250 ml and flat walls(T75-cell culture flasks). About 10 times×10⁶ cells were transferredinto each flasks and were each filled up with 20 ml of the aboveindicated nutrient medium. The determination of this cell number for theexact dosing per flask was carried out according to known procedures,cf. Hay R. J., “Cell Quantification and Characterization” in Methods ofTissue Engineering, Academic Press (2002), Chapter 4, pages 55-84.

[0316] The cell culture flasks were incubated in an incubator at 37° C.for 6 days. After 24 hours, the cells settled at the bottom of theflasks. The supernatant was removed every second day and the flasks wereeach filled with 20 ml fresh nutrient medium.

[0317] On day 6, the flasks were rinsed twice with 10 ml PBS each, afterthe nutrient medium had previously been pipetted off from the flasks.Hereby, all cells were removed, which did not adhere to the bottom ofthe flasks. The cells growing adhere to the bottom of the flasks weresubsequently removed from the bottom of the flasks with a sterile cellscraper. The separated cells were now removed from the flasks by rinsingwith PBS and were pooled in a 50 ml Falcon tube and were centrifuged at1800 rpm for 10 minutes. Thereafter, the supernatant was discarded andthe sediment was resuspended in fresh RPMI 1640 medium (2 ml/10⁵ cells).

[0318] This cell suspension could be used directly for differentiatinginto various target cells.

[0319] Alternatively, the cells were mixed with DMSO/FCS as freezingmedium after centrifugation and were deep-frozen at a concentration of10⁶/ml.

[0320] The freezing medium contained 95% FCS and 5% DMSO. About 10 cellswere taken up in 1 ml of the medium and were cooled following thesubsequent steps:

[0321] 30 minutes on ice;

[0322] 2 hours at −20° C. in precooled styropor box;

[0323] 24 hours at −80° C. in styropor;

[0324] stored in tubes in liquid nitrogen (N₂) at −180° C.

1. A process for the production of dedifferentiated, programmable stemcells of human monocytic origin, comprising: a) isolating monocytes fromhuman blood; b) propagating the monocytes in a culture medium, whichcontains cellular growth factor M-CSF; c) simultaneously cultivating themonocytes with or subsequently to step b) in a culture medium comprisingIL-3; and d) obtaining human adult dedifferentiated programmable stemcells by separating from culture medium.
 2. The process according toclaim 1, wherein said culture medium comprising IL-3 further comprises amercapto compound.
 3. The process according to claim 2, wherein saidmercapto compound has at least one carbon group bonded to the sulfur,and wherein hydrocarbon groups may be substituted with one or morefunctional groups.
 4. The process according to claim 2, wherein saidmercapto compound is 2-mercaptoethanol or dimethylsulfoxide.
 5. Theprocess according to claim 1, further comprising contacting the cellswith a biologically acceptable organic solvent.
 6. The process accordingto claim 2, further comprising contacting the cells with a biologicallyacceptable organic solvent.
 7. The process according to claim 5, whereinsaid biologically acceptable organic solvent is added after saidcultivation of said monocytes in a culture medium containing IL-3 butbefore said separating of said human adult dedifferentiated programmablestem cells from said culture medium.
 8. The process according to claim7, wherein said biologically acceptable organic solvent is an alcoholwith 1-4 carbon atoms.
 9. The process according to claim 8, wherein saidbiologically acceptable alcohol is ethanol.
 10. The process according toclaim 7, wherein said cultivated monocyte cells are brought into contactwith the vapor phase of the biologically acceptable organic solvent. 11.The process according to claim 1, further comprising suspending saidcultivated monocyte cells in a suitable cell culture medium subsequentto step d).
 12. The process according to claim 11, wherein saidsuspension medium is RPMI or DMEM.
 13. The process according to claim11, wherein said suspension medium comprises a cytokine or LIF.
 14. Theprocess according to claim 11, wherein said cultivated monocyte cellsare deep frozen.
 15. The process according to claim 14, wherein saidsuspension medium comprises a cytokine or LIF.
 16. A dedifferentiated,programmable stem cell of human monocytic origin, wherein said cell ischaracterized by exhibiting a CD14 antigen and a CD123 antigen.
 17. Astem cell of claim 16, further comprising a transfected gene.
 18. Adedifferentiated, programmable stem cell of human monocytic origin,wherein said cell is characterized by exhibiting a CD14 antigen and aCD135 antigen.
 19. A stem cell of claim 18, further comprising atransfected gene.
 20. A dedifferentiated, programmable stem cell ofhuman monocytic origin, wherein said cell is characterized by exhibitinga CD14 antigen, a CD123 antigen and a CD135 antigen.
 21. A stem cell ofclaim 20, further comprising a transfected gene.
 22. A dedifferentiated,programmable stem cell of human monocytic origin manufactured by aprocess comprising: a) isolating monocytes from human blood; b)propagating monocytes in a culture medium, which contains cellulargrowth factor M-CSF; c) simultaneously cultivating monocytes with orsubsequently to step b) in a culture medium comprising IL-3; and d)obtaining human adult dedifferentiated programmable stem cells byseparating from culture medium.
 23. A pharmaceutical compositioncomprising a dedifferentiated, programmable stem cell of human monocyticorigin, wherein said cell is characterized by exhibiting a CD14 antigenand a CD135 antigen.
 24. A pharmaceutical composition comprising adedifferentiated, programmable stem cell of human monocytic origin,wherein said cell is characterized by exhibiting a CD14 antigen and aCD123 antigen.
 25. A pharmaceutical composition comprising adedifferentiated, programmable stem cell of human monocytic origin,wherein said cell is characterized by exhibiting a CD14 antigen, a CD123antigen and a CD135 antigen.
 26. A method of producing target cells fromdedifferentiated, programmable stem cells of human monocytic origincomprising: a) obtaining desired target cells from a target tissue; b)incubating said desired target cells in a suitable culture medium; andc) providing supernatant from said culture medium after incubation withsaid desired target cells to dedifferentiated, programmable stem cellsof human monocytic origin that are characterized by exhibiting a CD14and a CD135 antigen to differentiate said stem cells of human monocyticorigin into target cells.
 27. A method according to claim 26, whereinsaid stem cells of human monocytic origin are differentiated intoadipocytes, neurons and glia cells, endothelial cells, keratinocytes,hepatocytes or islet cells.
 28. A method of producing target cells fromdedifferentiated, programmable stem cells of human monocytic origincomprising: a) obtaining desired target cells from a target tissue; b)incubating said desired target cells in a suitable culture medium; andc) providing supernatant from said culture medium after incubation withsaid desired target cells to dedifferentiated, programmable stem cellsof human monocytic origin that are characterized by exhibiting a CD14antigen and a CD123 antigen to differentiate said stem cells of humanmonocytic origin into target cells.
 29. A method according to claim 28,wherein said stem cells of human monocytic origin are differentiatedinto adipocytes, neurons and glia cells, endothelial cells,keratinocytes, hepatocytes or islet cells.
 30. A method of producingtarget cells from dedifferentiated, programmable stem cells of humanmonocytic origin comprising: a) obtaining desired target cells from atarget tissue; b) incubating said desired target cells in a suitableculture medium; and c) providing supernatant from said culture mediumafter incubation with said desired target cells to dedifferentiated,programmable stem cells of human monocytic origin that are characterizedby exhibiting a CD14 antigen, a CD123 antigen and a CD135 antigen todifferentiate said stem cells of human monocytic origin into targetcells.
 31. A method according to claim 30, wherein said stem cells ofhuman monocytic origin are differentiated into adipocytes, neurons andglia cells, endothelial cells, keratinocytes, hepatocytes or isletcells.
 32. A dedifferentiated, programmable stem cell of human monocyticorigin, wherein said cell is characterized by the membrane associatedmonocyte-specific surface antigen CD14 and at least one pluripotencymarker selected from the group consisting of CD117, CD123 and CD135. 33.A dedifferentiated, programmable stem cell according to claim 32,wherein said dedifferentiated, programmable stem cell is transfectedwith one or more genes.
 34. A dedifferentiated, programmable stem cellpreparation according to claim 32 in a suitable medium.